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

The automation of liquid-handling routines offers great potential for fast, reproducible, and labor-reduced biomaterial fabrication but also requires the development of special protocols. Competitive systems demand for a high degree in miniaturization and parallelization while maintaining flexibility regarding the experimental design. Today, there are only a few possibilities for automated fabrication of biomaterials inside multiwell plates. We have previously demonstrated that streptavidin-based biomimetic platforms can be employed to study cellular behaviors on biomimetic surfaces. So far, these self-assembled materials were made by stepwise assembly of the components using manual pipetting. In this work, we introduce for the first time a fully automated and adaptable workflow to functionalize glass-bottom multiwell plates with customized biomimetic platforms deposited in single wells using a liquid-handling robot. We then characterize the cell response using automated image acquisition and subsequent analysis. Furthermore, the molecular surface density of the biomimetic platforms was characterized in situ using fluorescence-based image correlation spectroscopy. These measurements were in agreement with standard ex situ spectroscopic ellipsometry measurements. Due to automation, we could do a proof of concept to study the effect of heparan sulfate on the bioactivity of bone morphogenetic proteins on myoblast cells, using four different bone morphogenetic proteins (BMPs) (2, 4, 6, and 7) in parallel, at five increasing concentrations. Using such an automated self-assembly of biomimetic materials, it may be envisioned to further investigate the role of a large variety of extracellular matrix (ECM) components and growth factors on cell signaling.

Combining Fluorescence Fluctuations and Photobleaching to Quantify Surface Density.

We have established a self-calibrated method, called pbFFS for photobleaching fluctuation fluorescence spectroscopy, which aims to characterize molecules or particles labeled with an unknown distribution of fluorophores. Using photobleaching as a control parameter, pbFFS provides information on the distribution of fluorescent labels and a reliable estimation of the absolute density or concentration of these molecules. We present a complete theoretical derivation of the pbFFS approach and experimentally apply it to measure the surface density of a monolayer of fluorescently tagged streptavidin molecules, which can be used as a base platform for biomimetic systems. The surface density measured by pbFFS is consistent with the results of spectroscopic ellipsometry, a standard surface technique. However, pbFFS has two main advantages: it enables in situ characterization (no dedicated substrates are required) and can be applied to low masses of adsorbed molecules, which we demonstrate here by quantifying the density of biotin-Atto molecules that bind to the streptavidin layer. In addition to molecules immobilized on a surface, we also applied pbFFS to molecules diffusing in solution, to confirm the distribution of fluorescent labels found on a surface. Hence, pbFFS provides a set of tools for investigating the molecules labeled with a variable number of fluorophores, with the aim of quantifying either the number of molecules or the distribution of fluorescent labels, the latter case being especially relevant for oligomerization studies.

Proton therapy monitoring: spatiotemporal emission reconstruction with prompt gamma timing and implementation with PET detectors.

Objective. In this study we introduce spatiotemporal emission reconstruction prompt gamma timing (SER-PGT), a new method to directly reconstruct the prompt photon emission in the space and time domains inside the patient in proton therapy.Approach. SER-PGT is based on the numerical optimisation of a multidimensional likelihood function, followed by a post-processing of the results. The current approach relies on a specific implementation of the maximum-likelihood expectation maximisation algorithm. The robustness of the method is guaranteed by the complete absence of any information about the target composition in the algorithm.Main results. Accurate Monte Carlo simulations indicate a range resolution of about 0.5 cm (standard deviation) when considering 107primary protons impinging on an homogeneous phantom. Preliminary results on an anthropomorphic phantom are also reported.Significance. By showing the feasibility for the reconstruction of the primary particle range using PET detectors, this study provides significant basis for the development of an hybrid in-beam PET and prompt photon device.

Heparan sulfate co-immobilized with cRGD ligands and BMP2 on biomimetic platforms promotes BMP2-mediated osteogenic differentiation.

The chemical and physical properties of the extracellular matrix (ECM) are known to be fundamental for regulating growth factor bioactivity. The role of heparan sulfate (HS), a glycosaminoglycan, and of cell adhesion proteins (containing the cyclic RGD (cRGD) ligands) on bone morphogenetic protein 2 (BMP2)-mediated osteogenic differentiation has not been fully explored. In particular, it is not known whether and how their effects can be potentiated when they are presented in controlled close proximity, as in the ECM. Here, we developed streptavidin platforms to mimic selective aspects of the in vivo presentation of cRGD, HS and BMP2, with a nanoscale-control of their surface density and orientation to study cell adhesion and osteogenic differentiation. We showed that whereas a controlled increase in cRGD surface concentration upregulated BMP2 signaling due to ß3 integrin recruitment, silencing either ß1 or ß3 integrins negatively affected BMP2-mediated phosphorylation of SMAD1/5/9 and alkaline phosphatase expression. Furthermore, the presence of adsorbed BMP2 promoted cellular adhesion at very low cRGD concentrations. Finally, we proved that HS co-immobilized with cRGD both sustained BMP2 signaling and enhanced osteogenic differentiation compared to BMP2 directly immobilized on streptavidin, even with a low cRGD surface concentration. Altogether, our results show that HS facilitated and sustained the synergy between BMP2 and integrin pathways and that the co-immobilization of HS and cRGD peptides optimised BMP2-mediated osteogenic differentiation. Statement of significance The growth factor BMP2 is used to treat large bone defects. Previous studies have shown that the presentation of BMP2 via extracellular matrix molecules, such as heparan sulfate (HS), can upregulate BMP2 signaling. The potential advantages of dose reduction and local specificity have stimulated interest in further investigations into biomimetic approaches. We designed a streptavidin model surface eligible for immobilizing tunable amounts of molecules from the extracellular space, such as HS, adhesion motifs (cyclic RGD) and BMP2. By studying cellular adhesion, BMP2 bioactivity and its osteogenic potential we reveal the combined effect of integrins, HS and BMP2, which contribute in answering fundamental questions regarding cell-matrix interaction.