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Advancing RTHS methods to readily handle multi-dimensional problems has great potential for enabling more advanced testing and synergistically using existing laboratory facilities that have the capacity for such experimentation. However, the high internal coupling between hydraulics actuators and the nonlinear kinematics escalates the complexity of actuator control and boundary condition tracking. To enable researchers in the RTHS community to develop and compare advanced control algorithms, this paper proposes a benchmark control problem for a multi-axial real-time hybrid simulation (maRTHS) and presents its definition and implementation on a steel frame excited by seismic loads at the base. The benchmark problem enables the development and validation of control techniques for tracking both translation and rotation degrees of freedom of a plant that consists of a steel frame, two hydraulic actuators, and a steel coupler with high stiffness that couples the axial displacements of the hydraulic actuators resulting in the required motion of the frame node. In this investigation, the different components of this benchmark were developed, tested, and a set of maRTHS were conducted to demonstrate its feasibility in order to provide a realistic virtual platform. To offer flexibility in the control design process, experimental data for identification purposes, finite element models for the reference structure, numerical, and physical substructure, and plant models with model uncertainties are provided. Also, a sample example of an RTHS design based on a linear quadratic Gaussian controller is included as part of a computational code package, which facilitates the exploration of the tradeoff between robustness and performance of tracking control designs. The goals of this benchmark are to: extend existing control or develop new control techniques; provide a computational tool for investigation of the challenging aspects of maRTHS; encourage a transition to multiple actuator RTHS scenarios; and make available a challenging problem for new researchers to investigate maRTHS approaches. We believe that this benchmark problem will encourage the advancing of the next-generation of controllers for more realistic RTHS methods.

Mimicking bone extracellular matrix (ECM) is paramount to develop novel biomaterials for bone tissue engineering. In this regard, the combination of integrin-binding ligands together with osteogenic peptides represents a powerful approach to recapitulate the healing microenvironment of bone. In the present work, we designed polyethylene glycol (PEG)-based hydrogels functionalized with cell instructive multifunctional biomimetic peptides (either with cyclic RGD-DWIVA or cyclic RGD-cyclic DWIVA) and cross-linked with matrix metalloproteinases (MMPs)-degradable sequences to enable dynamic enzymatic biodegradation and cell spreading and differentiation. The analysis of the intrinsic properties of the hydrogel revealed relevant mechanical properties, porosity, swelling and degradability to engineer hydrogels for bone tissue engineering. Moreover, the engineered hydrogels were able to promote human mesenchymal stem cells (MSCs) spreading and significantly improve their osteogenic differentiation. Thus, these novel hydrogels could be a promising candidate for applications in bone tissue engineering, such as acellular systems to be implanted and regenerate bone or in stem cells therapy.

The tumor microenvironment plays an important role in cancer development and the use of 3D in vitro systems that decouple different elements of this microenvironment is critical for the study of cancer progression. In neuroblastoma (NB), vitronectin (VN), an extracellular matrix protein, has been linked to poor prognosis and appears as a promising therapeutic target. Here, we developed hydrogels that incorporate VN into 3D polyethylene glycol (PEG) hydrogel networks to recapitulate the native NB microenvironment. The stiffness of the VN/PEG hydrogels was modulated to be comparable to the in vivo values reported for NB tissue samples. We used SK-N-BE (2) NB cells to demonstrate that PEGylated VN promotes cell adhesion as the native protein does. Furthermore, the PEGylation of VN allows its crosslinking into the hydrogel network, providing VN retention within the hydrogels that support viable cells in 3D. Confocal imaging and ELISA assays indicate that cells secrete VN also in the hydrogels and continue to reorganize their 3D environment. Overall, the 3D VN-based PEG hydrogels recapitulate the complexity of the native tumor extracellular matrix, showing that VN-cell interaction plays a key role in NB aggressiveness, and that VN could potentially be targeted in preclinical drug studies performed on the presented hydrogels.

Embryonic stem cells (ESCs) possess remarkable abilities, as they can differentiate into all cell types (pluripotency) and be self-renewing, giving rise to two identical cells. These characteristics make ESCs a powerful research tool in fundamental embryogenesis as well as candidates for use in regenerative medicine. Significant efforts have been devoted to developing protocols to control ESC fate, including soluble and complex cocktails of growth factors and small molecules seeking to activate/inhibit key signaling pathways for the maintenance of pluripotency states or activate differentiation. Here we describe a novel method for the effective maintenance of mouse ESCs, avoiding the supplementation of complex inhibitory cocktails or cytokines, e.g., LIF. We show that the addition of zinc to ESC cultures leads to a stable pluripotent state that shares biochemical, transcriptional and karyotypic features with the classical LIF treatment. We demonstrate for the first time that ESCs maintained in long-term cultures with added zinc, are capable of sustaining a stable ESCs pluripotent phenotype, as well as differentiating efficiently upon external stimulation. We show that zinc promotes long-term ESC self-renewal (>30 days) via activation of ZIP7 and AKT signaling pathways. Furthermore, the combination of zinc with LIF results in a synergistic effect that enhances LIF effects, increases AKT and STAT3 activity, promotes the expression of pluripotency regulators and avoids the expression of differentiation markers.