gms | German Medical Science

Objectives: Demographics are changing, resulting in older patients, thus increasing the prevalence of degenerative musculoskeletal conditions, such as osteoporosis, and their complications, such as delayed and non-union healing of fractures. In this context we published the phenotype of the integrinα2 deficient (Itga2^-/-) mouse, which showed reduced age-related bone deterioration and faster fracture healing. A key ligand of integrinα2 (Itga2) is collagen I, the most abundant protein in bone. Itga2 may act as a "collagen sensor", since Itga2^-/- osteoblasts show higher collagen I synthesis and faster differentiation (earlier calcium and collagen deposition).

We hypothesised that the pharmacological down-regulation of Itga2 expression could be used to accelerate osteoblast differentiation and improve fracture healing. Therefore, we investigated E7820 and Ropivacaine, which have been shown to inhibit Itga2 expression on other cell types.

Methods: We used the well-established pre-osteoblast MC3T3 cell line throughout this investigation. Cytotoxicity was investigated using an MTT-assay. The expression of Itga2 on the mRNA level was assayed using qRT-PCR and the Itga2 protein abundance was examined by quantitative fluorescent western blot (qf-WB) or by In-Cell-Western (ICW) analysis. Phorbol-12-myristate-13-acetate (PMA) was used as a control. Osteoblastic differentiation was assessed using alizarin red staining of calcium deposition and total collagen deposition.

Results and conclusion: The MTT-assay showed a dose-dependent cytotoxicity of E7820 and Ropivacaine with half maximal inhibitory concentrations (EC₅₀) of 0.4μg/ml and 0.4 mg/ml respectively after 3 days of incubation. No significant change in absorption was detected up to 100 ng/ml and 0.01 mg/ml respectively. 50 ng/ml E7820 and 0.1 and 0.01 mg/ml Ropivacaine were used for further assays.

Surprisingly, E7820 induced a dose dependent increase in Itga2 expression and Ropivacaine did not effect a change in Itga2 expression. The control using PMA showed the expected increase in Itga2 expression. Neither of the trial substances accelerated osteoblast differentiation with respect to calcium deposition and E7820 did not affect total collagen deposition.

This data shows that E7820 and Ropivacaine have aberrant activity in MC3T3 cells, since they do not inhibit Itga2 expression, which stands in contrast to previously published data from other cell lines. It is currently not clear, if this is a cell line specific effect or a general phenomenon in osteoblastic cells. Investigation of the substances in other osteoblastic cell lines or primary osteoblasts would be needed to answer this question.

Alternatively, approaches that directly modulate the process of "collagen sensing“ by Itga2, for example disintegrins, such as Rhodocetin, or blocking antibodies, which inhibit Itga2 binding to collagen I, could be used to further investigate the Itga2^-/- phenotype and potentially leverage it to treat osteoporosis and to accelerate fracture healing.