Differential bioactivity of four BMP-family members as function of biomaterial stiffness

Abstract

Whereas soft biomaterial is not able to induce cell spreading, BMP-2 presented by a soft film has been described to be sufficient to trigger cell spreading, migration and downstream BMP-2 signaling. Based on thin polyelectrolyte films of controlled stiffness, we investigated whether the presentation of four BMP members (2, 4, 7, 9) in a matrix-bound manner may differentially impact cell adhesion and bone differentiation of skeletal progenitors. We performed high content and automated screening of cellular responses, including cell number, cell spreading area, SMAD phosphorylation and alkaline phosphatase activity. The basolateral presentation of the different BMPs allowed us to discriminate the specificity of cellular response and the role of BMP receptors type I, type II, as well as three β integrins, in a BMP type and stiffness-dependent manner.

Automated Fabrication of Streptavidin-Based Self-assembled Materials for High-Content Analysis of Cellular Response to Growth Factors

“…Sales et al also compared concentrations of matrix-bound BMP2, 4, and 7 to each other and found higher SMAD1/5/9 phosphorylation induced by BMP2 compared to BMP4 and BMP7 at low concentrations. 21 However, at higher concentrations, BMP7 was inducing the highest signal among all. Due to the novel workflow here reported, it will be possible to perform a systematic study of the effect of different GFs and different GAGs on cellular signaling.…”

“…Cells were imaged using InCell Analyzer 2500 using the 20× objective on three channels. Images were further analyzed with the automated image analysis software InCarta (Molecular Devices, San Jose, California), as previously described: 21 wells, and experiments were repeated in three biological replicates. Means were tested for statistical significance between different conditions with the nonparametric Mann−Whitney test with p = ≤ 0.05 and based on at least three biological replicates.…”

“…Sales et al further automated the workflow of this system by applying automated microscopy and automated image analysis to perform a highcontent screening of cell adhesion and early cell differentiation to four BMPs simultaneously. 21 Eggert et al recently developed the automated fabrication of hydrogels at the single-well level. 23 To our knowledge, this approach has never been applied to self-assembled materials made of several combinations of deposited layers.…”

“…7 We quantified SMAD1/5/9 phosphorylation in BMP-responsive C2C12 mouse myoblasts. 21,40,41 ■ METHODS Buffers and Molecules. 10 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (Hepes) and 150 mN NaCl buffer (Sigma-Aldrich, Saint-Quentin Fallavier, France), pH 7.2, named hereafter Hepes, was used for dilution and rinsing of all molecules if not further specified.…”

High-throughput measurements of bone morphogenetic protein/bone morphogenetic protein receptor interactions using biolayer interferometry

“…Indeed, previous data in our team show an expression of ALK5 in BMP-responsive cells notably C2C12 skeletal muscle cells and human periosteum derived stem cells. 64 Moreover, it is reported that ALK5 interacts with ALK1 and inhibits BMP signaling mediated by ALK1 in the growth plate of cartilage. 65 Also, BMP-2 appeared to induce complex formation between ALK3 and ALK5 in cancer cells.…”