Multiscale-Engineered Muscle Constructs: PEG Hydrogel Micro-Patterning on an Electrospun PCL Mat Functionalized with Gold Nanoparticles.

The development of new, viable, and functional engineered tissue is a complex and challenging task. Skeletal muscle constructs have specific requirements as cells are sensitive to the stiffness, geometry of the materials, and biological micro-environment. The aim of this study was thus to design and characterize a multi-scale scaffold and to evaluate it regarding the differentiation process of C2C12 skeletal myoblasts. The significance of the work lies in the microfabrication of lines of polyethylene glycol, on poly(ε-caprolactone) nanofiber sheets obtained using the electrospinning process, coated or not with gold nanoparticles to act as a potential substrate for electrical stimulation. The differentiation of C2C12 cells was studied over a period of seven days and quantified through both expression of specific genes, and analysis of the myotubes' alignment and length using confocal microscopy. We demonstrated that our multiscale bio-construct presented tunable mechanical properties and supported the different stages skeletal muscle, as well as improving the parallel orientation of the myotubes with a variation of less than 15°. These scaffolds showed the ability of sustained myogenic differentiation by enhancing the organization of reconstructed skeletal muscle. Moreover, they may be suitable for applications in mechanical and electrical stimulation to mimic the muscle's physiological functions.

Multi-Sensor Origami Platform: A Customizable System for Obtaining Spatiotemporally Precise Functional Readouts in 3D Models.

Bioprinting technology offers unprecedented opportunities to construct in vitro tissue models that recapitulate the 3D morphology and functionality of native tissue. Yet, it remains difficult to obtain adequate functional readouts from such models. In particular, it is challenging to position sensors in desired locations within pre-fabricated 3D bioprinted structures. At the same time, bioprinting tissue directly onto a sensing device is not feasible due to interference with the printer head. As such, a multi-sensing platform inspired by origami that overcomes these challenges by "folding" around a separately fabricated 3D tissue structure is proposed, allowing for the insertion of electrodes into precise locations, which are custom-defined using computer-aided-design software. The multi-sensing origami platform (MSOP) can be connected to a commercial multi-electrode array (MEA) system for data-acquisition and processing. To demonstrate the platform, how integrated 3D MEA electrodes can record neuronal electrical activity in a 3D model of a neurovascular unit is shown. The MSOP also enables a microvascular endothelial network to be cultured separately and integrated with the 3D tissue structure. Accordingly, how impedance-based sensors in the platform can measure endothelial barrier function is shown. It is further demonstrated the device's versatility by using it to measure neuronal activity in brain organoids.

Super-Resolution-Chip: an in-vitro platform that enables super-resolution microscopy of co-cultures and 3D systems.

The development of organs-on-a-chip platforms has revolutionized in-vitro cellular culture by allowing cells to be grown in an environment that better mimics human physiology. However, there is still a challenge in integrating those platforms with advanced imaging technology. This is extremely important when we want to study molecular changes and subcellular processes on the level of a single molecule using super-resolution microscopy (SRM), which has a resolution beyond the diffraction limit of light. Currently, existing platforms that include SRM have certain limitations, either as they only support 2D monocultures, without flow or as they demand a lot of production and handling. In this study, we developed a Super-Res-Chip platform, consisting of a 3D-printed chip and a porous membrane, that could be used to co-culture cells in close proximity either in 2D or in 3D while allowing SRM on both sides of the membrane. To demonstrate the functionality of the device, we co-cultured in endothelial and epithelial cells and used direct stochastic optical reconstruction microscopy (dSTORM) to investigate how glioblastoma cells affect the expression of the gap-junction protein Connexin43 in endothelial cells grown in 2D and in 3D. Cluster analysis of Connexin43 distribution revealed no difference in the number of clusters, their size, or radii, but did identify differences in their density. Furthermore, the spatial resolution was high also when the cells were imaged through the membrane (20-30 nm for x-y) and 10-20 nm when imaged directly both for 2D and 3D conditions. Overall, this chip allows to characterize of complex cellular processes on a molecular scale in an easy manner and improved the capacity for imaging in a single molecule resolution complex cellular organization.

Traumatic Brain Injury in a Well: A Modular Three-Dimensional Printed Tool for Inducing Traumatic Brain Injury In vitro.

Traumatic brain injury (TBI) is a major health problem that affects millions of persons worldwide every year among all age groups, mainly young children, and elderly persons. It is the leading cause of death for children under the age of 16 and is highly correlated with a variety of neuronal disorders, such as epilepsy, and neurodegenerative disease, such as Alzheimer's disease or amyotrophic lateral sclerosis. Over the past few decades, our comprehension of the molecular pathway of TBI has improved, yet despite being a major public health issue, there is currently no U.S. Food and Drug Administration-approved treatment for TBI, and a gap remains between these advances and their application to the clinical treatment of TBI. One of the major hurdles for pushing TBI research forward is the accessibility of TBI models and tools. Most of the TBI models require costume-made, complex, and expensive equipment, which often requires special knowledge to operate. In this study, we present a modular, three-dimensional printed TBI induction device, which induces, by the pulse of a pressure shock, a TBI-like injury on any standard cell-culture tool. Moreover, we demonstrate that our device can be used on multiple systems and cell types and can induce repetitive TBIs, which is very common in clinical TBI. Further, we demonstrate that our platform can recapitulate the hallmarks of TBI, which include cell death, decrease in neuronal functionality, axonal swelling (for neurons), and increase permeability (for endothelium). In addition, in view of the continued discussion on the need, benefits, and ethics of the use of animals in scientific research, this in vitro, high-throughput platform will make TBI research more accessible to other labs that prefer to avoid the use of animals yet are interested in this field. We believe that this will enable us to push the field forward and facilitate/accelerate the availability of novel treatments.

Inducing Mechanical Stimuli to Tissues Grown on a Magnetic Gel Allows Deconvoluting the Forces Leading to Traumatic Brain Injury.

Traumatic brain injury (TBI), which is characterized by damage to the brain resulting from a sudden traumatic event, is a major cause of death and disability worldwide. It has short- and long-term effects, including neuroinflammation, cognitive deficits, and depression. TBI consists of multiple steps that may sometimes have opposing effects or mechanisms, making it challenging to investigate and translate new knowledge into effective therapies. In order to better understand and address the underlying mechanisms of TBI, we have developed an in vitro platform that allows dynamic simulation of TBI conditions by applying external magnetic forces to induce acceleration and deceleration injury, which is often observed in human TBI. Endothelial and neuron-like cells were successfully grown on magnetic gels and applied to the platform. Both cell types showed an instant response to the TBI model, but the endothelial cells were able to recover quickly-in contrast to the neuron-like cells. In conclusion, the presented in vitro model mimics the mechanical processes of acceleration/deceleration injury involved in TBI and will be a valuable resource for further research on brain injury.

Monitoring mechanical stimulation for optimal tendon tissue engineering: A mechanical and biological multiscale study.

To understand the effect of mechanical stimulation on cell response, bone marrow stromal cells were cultured on electrospun scaffolds under two distinct mechanical conditions (static and dynamic). Comparison between initial and final mechanical and biological properties of the cell-constructs were conducted over 14 days for both culturing conditions. As a result, mechanically stimulated constructs, in contrast to their static counterparts, showed evident mechanical-induced cell orientation, an effective aligned collagen and tenomodulin extracellular matrix. This orientation provides clues on the importance of mechanical stimulation to induce a tendon-like differentiation. In addition, cell and collagen orientation lead to enhanced storage modulus observed under dynamic stimulation. Altogether mechanical stimulation lead to (a) cell and matrix orientation through the sense of the stretch and (b) a dominant elastic response in the cell-constructs with a minor contribution of the viscosity in the global mechanical behavior. Such a correlation could help in further studies to better understand the effect of mechanical stimulation in tissue engineering.

Biomaterials in Tendon and Skeletal Muscle Tissue Engineering: Current Trends and Challenges.

Tissue engineering is a promising approach to repair tendon and muscle when natural healing fails. Biohybrid constructs obtained after cells’ seeding and culture in dedicated scaffolds have indeed been considered as relevant tools for mimicking native tissue, leading to a better integration in vivo. They can also be employed to perform advanced in vitro studies to model the cell differentiation or regeneration processes. In this review, we report and analyze the different solutions proposed in literature, for the reconstruction of tendon, muscle, and the myotendinous junction. They classically rely on the three pillars of tissue engineering, i.e., cells, biomaterials and environment (both chemical and physical stimuli). We have chosen to present biomimetic or bioinspired strategies based on understanding of the native tissue structure/functions/properties of the tissue of interest. For each tissue, we sorted the relevant publications according to an increasing degree of complexity in the materials’ shape or manufacture. We present their biological and mechanical performances, observed in vitro and in vivo when available. Although there is no consensus for a gold standard technique to reconstruct these musculo-skeletal tissues, the reader can find different ways to progress in the field and to understand the recent history in the choice of materials, from collagen to polymer-based matrices.