Benjamin A. Byers

Enhanced expression of the osteoblastic phenotype on substrates that modulate fibronectin conformation and integrin receptor binding

Integrins represent the primary mechanism of cell-extracellular matrix interactions and control cell morphology, proliferation, and differentiation. We have previously shown that substrate-dependent modulation of adsorbed fibronectin (Fn) conformation alters alpha5beta1 integrin binding to Fn and directs C2C12 myoblast proliferation and differentiation (Mol. Biol. Cell 10 (1999) 785). The model substrates used in these experiments were bacteriological (untreated) polystyrene (B), tissue culture polystyrene (T), and type-I collagen-coated T (C). In the present study, we examined MC3T3-EI osteoblast-like cell differentiation on Fn-coated B, T, and C substrates. Immunofluorescence staining revealed substrate-dependent differences in integrin alpha5beta1 binding and clustering into focal adhesions (C > T > B), consistent with our previous integrin binding analysis. Alkaline phosphatase activity and matrix mineralization showed substrate-dependent differences (C > T > B, p < 0.05). Similar trends were observed for alkaline phosphatase, osteocalcin, and bone sialoprotein gene expression. Blocking experiments with antibodies directed against Fn completely inhibited matrix mineralization on Fn-coated C, indicating that Fn is critical to expression of the osteoblastic phenotype on this extracellular matrix component. These substrate-dependent differences in osteoblast differentiation correlated with differences in alpha5beta1 binding, suggesting that these differences arise from substrate modulation of integrin-matrix interactions. Substrate-dependent modulation of cell function may provide a versatile mechanism to control cell responses in numerous biomedical applications.

Cell-Type-Dependent Up-Regulation of In Vitro Mineralization After Overexpression of the Osteoblast-Specific Transcription Factor Runx2/Cbfa1†

Functional expression of the transcriptional activator Runx2/Cbfa1 is essential for osteoblastic differentiation and bone formation and maintenance. Forced expression of Runx2 in nonosteoblastic cells induces expression of osteoblast‐specific genes, but the effects of Runx2 overexpression on in vitro matrix mineralization have not been determined. To examine whether exogenous Runx2 expression is sufficient to direct in vitro mineralization, we investigated sustained expression of Runx2 in nonosteoblastic and osteoblast‐like cell lines using retroviral gene delivery. As expected, forced expression of Runx2 induced several osteoblast‐specific genes in NIH3T3 and C3H10T1/2 fibroblasts and up‐regulated expression in MC3T3‐E1 immature osteoblast‐like cells. However, Runx2 expression enhanced matrix mineralization in a cell‐type‐dependent manner. NIH3T3 and IMR‐90 fibroblasts overexpressing Runx2 did not produce a mineralized matrix, indicating that forced expression of Runx2 in these nonosteogenic cell lines is not sufficient to direct in vitro mineralization. Consistent with the pluripotent nature of the cell line, a fraction (25%) of Runx2‐expressing C3H10T1/2 fibroblast cultures produced mineralized nodules in a viral supernatant‐dependent manner. Notably, bone sialoprotein (BSP) gene expression was detected at significantly higher levels in mineralizing Runx2‐infected C3H10T1/2 cells compared with Runx2‐expressing cultures which did not mineralize. Treatment of Runx2‐infected C3H10T1/2 cultures with dexamethasone enhanced osteoblastic phenotype expression, inducing low levels of mineralization independent of viral supernatant. Finally, Runx2 overexpression in immature osteoblast‐like MC3T3‐E1 cells resulted in acceleration and robust up‐regulation of matrix mineralization compared with controls. These results suggest that, although functional Runx2 is essential to multiple osteoblast‐specific activities, in vitro matrix mineralization requires additional tissue‐specific cofactors, which supplement Runx2 activity.

Addressing cell-sourcing limitations with gene therapy

Genetic engineering with Run/spl times/2/Cbfa 1 for an alternative to biological grafts. Forced Runx2 expression via retroviral gene delivery promotes osteoblastic differentiation and mineralization in bone marrow stromal cells and stimulates conversion of myoblasts and dermal fibroblasts into a mineralizing osteoblastic phenotype. This genetic engineering strategy provides a promising approach to overcome cell-sourcing limitations in bone tissue engineering.

Genetically engineered non-osteoblastic cells expressing a transcriptional activator of osteoblast differentiation

In order to investigate alternative methods for the development of a tissue-engineered bone graft, this research focuses on forced expression of Osf2, an osteoblast-specific transcriptional activator, in 293 cells. Results indicate that, upon transfection, these non-osteoblastic cells express the Osf2 gene product and, therefore, maybe induced to express the osteoblastic phenotype.

Modulation of osteoblastic phenotype by substrate-dependent changes in fibronectin conformation

Adsorption of fibronectin onto untreated and tissue culture grade polystyrenes and collagen type I resulted in changes in the conformation of the adsorbed molecule. These changes in conformation altered the binding of specific integrins (/spl alpha//sub 3//spl beta//sub 1/) to fibronectin. This modulation in integrin binding controlled osteoblast-specific gene expression in human SaOs-2 osteoblast-like cells. The principle of controlling cell function by conformational changes in the extracellular matrix represents a powerful approach to engineer surfaces that elicit targeted cellular responses.