PI : dr. hab. Marco Costantini

In vitro tissue models are regarded as a critical component of future biomedical research. Using them, researchers have demonstrated a high capacity in mimicking both tissue-specific complex phenomena and revealing the interplay between physiological tissue structures and functions over the last two decades. Current in vitro tissue models, on the other hand, are mostly created using low-throughput approaches based on prior experience, intuition, and trial-and-error methodology. Such constraints result in the creation of valuable in vitro models, albeit far from optimal. The goal of this project is to address these limitations by creating a data-driven, high-throughput workflow that includes an innovative microfluidic-assisted extrusion bioprinting (µ-eBP) system supported by advanced image analysis tools for capturing key information about in vitro neo-tissue formation dynamics. The core scientific and technical objectives of the MYO-PATH project are: i) the progress towards a deeper understanding and control of cellular dynamic processes involved in the manufacturing of artificial tissues – specifically of skeletal muscle tissue; ii) the establishment of blueprints to obtain the highest tissue functionality and scalability potential and prioritize the best cost-effective experimental conditions; ii) the generation of a developmental atlas of artificial muscle morphogenesis in vitro. We intend to achieve this goal by developing new tools - hardware and software - for all stages of µ-eBP. A multiplexed and iterative screening and optimization approach will be used to obtain blueprint information. We plan to develop microfluidic modules that will allow us to address critical, currently unmet challenges in eBP, such as i) cell sedimentation mitigation in printer cartridges and ii) bioink multiplexing. Cellular dynamics will be captured using a variety of techniques such as bright-field, confocal, and optical coherence tomography. Advanced image analysis tools, including machine learning (ML)-based tools, will be used to analyse the resulting datasets. Notably, we propose developing quasi-live and offline image analysis tools to reliably assess the best performing conditions based on two indexes: an order parameter and a myo-index.

PI : dr. Nehar Celikkin

Skeletal muscle (SM) is a heterogeneous tissue consisting of large multinucleated muscle fibers interspersed with other cell types like adipocytes, satellite, and endothelial cells. Besides its primary function in movement, SM maintains homeostasis by regulating energy production in the body. One of its most striking physiological features to maintain homeostasis is its significant capacity to adapt to changed functional demands. Due to its plasticity, the SM responds to exercise and electrical stimulation; yet the underlying mechanisms are not fully elucidated. Indeed, high throughput analysis and real-time monitoring are required to reveal the underlying adaptation mechanism of SM; nevertheless, collecting a vast amount of biopsies and building up clinical studies with high precision is highly challenging and costly. To conduct these observations biomimicking in vitro models hold significant importance as they can be used as a representative element to study SM capacity of adaption to changed functional demands. Besides, such in vitro models can reveal the underlying mechanism in healthy developmental conditions and suggest how SM adapts under pathological conditions.

Regardless of the in vitro model and the stimuli protocol used, current in vitro SM studies are based on low-throughput approaches and previously published literature. Such constraints, combined with biological dynamics not being thoroughly captured during in vitro culture, result in valuable, however contradictory results.

This project addresses the current limitations by creating a high-throughput workflow that proposes an innovative approach for designing engineered muscle fascicles, capturing the real-time functionality and high throughput end-point proteomic response of engineered SM models to electro-mechanical stimuli.t

PI : mgr. Maria Celeste Tirelli

Nature demonstrates extraordinary abilities to create extraordinary complex structural-functional living materials with precise organization from the nano to the macro scale, a remarkable example can be the osteochondral (OC) region, which is the interface between hyaline cartilage and bone tissues. However, replicating this level of complexity in vitro remains a major challenge for tissue engineering and regenerative medicine. This project aims to address this challenge by developing a new approach for biofabrication, specifically targeting the creation of advanced materials that mimic the composition and architecture transitions found in the osteochondral regions. We plan to simplify and enhance the design and manufacturing process of 3D porous functionally graded hydrogels (pFGhs) that mimic the structural and functional organization of the OC region by utilizing novel bioactive inks and microfluidic printing heads in conjunction with a custom digital manufacturing platform. The interfaces between bone and cartilage exhibit a gradient in chemical and biological composition, as varying porosity ranges from a trabecular to a solid structure and gradient of cell type, which poses a challenge for inducing biomimetic differentiation of stem cells within a single culture system. To push the boundaries of current knowledge, one potential strategy is to enhance the bioactivity of matrices by formulating bioink containing functional-encoded peptides - which will be synthesized during the project - specifically targeting the differentiation of human mesenchymal stem cells (hMSCs) for bone/cartilage tissues and remodeling of the matrix. These bioinks will be used to produce pFGhs. Digital manufacturing offers numerous advantages, including rapid prototyping, accurate spatial deposition, repeatability, customization, and minimal macro-shape limitation. When combined with the capabilities of microfluidics, we can control the micro-architectural features and the composition of the resulting porous materials. The microfluidic printing head plays a vital role in this process as it comprises a passive micromixer and a flow-focusing junction. The micromixer homogenizes the streams of two continuous phases, while the flow-focusing junction generates droplets and controls their size. By combining the synthesis of novel bioink with 3D printing and microfluidics, we anticipate unprecedented control over local material properties, including pore size, pore connectivity, material composition, and biological activity. We firmly believe that the successful implementation of this project will unlock new technologies that facilitate the optimal reconstruction of interfacial tissue.