• Multimaterial
• Bioprinting
• Tissue Engineering
• Enthesis
• Multiscale
• Biofabrication

The human musculoskeletal system is a fascinating and intricate network of bones, muscles, joints, and connective tissues. The muscular system, responsible for generating the force required for movement, is a critical component. The skeletal system provides the structural framework for the body, and it has various bone types adapted to specific functions. The accessory structures, including joints, tendons, ligaments, bursae, and menisci, play a vital role in ensuring the integrity and function of the musculoskeletal system. Within these accessory structures, tendons and ligaments are specialized fibrous tissues that connect muscles-to-bones and bone-to bone, respectively. These connections are provided by the enthesis, a complex interface tissue. It allows for efficient force transmission and minimizes the risk of injuries. In human body, it possible to distinguish two types of entheses: fibrous and fibrocartilaginous. Fibrous entheses have collagen fibers attaching to the periosteum, creating a gradual transition for flexibility and minimal risk of injury. Fibrocartilaginous entheses contain four distinct zones, including a pure dense fibrous connective tissue, an uncalcified and a calcified fibrocartilage, separated by a tidemark, and finally a bone region. These zones are involved in stress absorption and force transfer, making the enthesis a dynamic and complex structure. The fibrocartilaginous enthesis holds particular fascination due to its complex organization but also its high susceptibility to injuries and the challenge represented by its reverse engineering. This research project aims to address the complex challenge of developing an enthesis-like scaffold with properties that faithfully mimic those of natural entheses. The study started by establishing a biofabrication protocol, a pivotal step in crafting a scaffold that could effectively combine the strength of bone and the elasticity of tendons and ligaments while replicating both macro- and nanoscale properties of natural entheses. In this frame, Tissue Engineering (TE) approaches can be considered as an added value. For this work, a combination of 3D printing and electrospinning technologies was employed. The selection of appropriate polymers played a key role in the development of the enthesis-like scaffold. Natural and synthetic polymers were methodically tested for biocompatibility using marrow-derived mesenchymal stem cells (BM-MSCs). Promising results were obtained, demonstrating the capability of these polymers to support cell adhesion and proliferation. The biofabrication protocol successfully recreated the gradient of physical properties within the enthesis-like scaffold. This novel process involved the direct 3D printing of PCL onto electrospun PLGA mats, allowing for the precise construction of a grid structure on the PLGA strips. The scaffold comprises three distinct regions able to replicate the enthesis bone, fibrocartilage, and tendon/ligament regions. An extensive characterization from the morphological, mechanical, and biological point of view was conducted to showcase all the scaffold features and its suitability for potential applications in bone-tendon interfaces. SEM image analysis revealed the scaffold multiscale and multimaterial properties, with each region showcasing distinct morphological features. The mechanical characterization was conducted to study the behavior of each region of the scaffold during a tensile test until fracture. Moreover, the strength of the interface between tendon-like and bone-like region was demonstrated. A preliminary biological study involving the differentiation testing of bone BM-MSCs on the enthesis scaffold demonstrated its potential for regenerative medicine applications. In the context of encouraging stem cell differentiation, a bioreactor system tailored for precise and effective tensile stimulation of enthesis scaffolds was developed. This system, incorporating fixed and mobile anchors along with a silicone insert, facilitated mechanical stretching to enhance cell infiltration, promote extracellular matrix deposition and alignment of collagen fibers. For a comprehensive biological validation, human mesenchymal stromal cells were used. The results demonstrated the scaffold's ability to support cell attachment, migration, and proliferation. Moreover, cyclic strain stimulation using the bioreactor was shown to enhance cell morphology and orientation, especially in the tendon-like region. An important development in this research project was the design of a biofabrication machine capable of producing multiscale structures in a single process, utilizing Melt Electrowriting (MEW) technology. This innovative machine opens the door to future advancements in enthesis engineering and musculoskeletal regenerative medicine. This research can represent a substantial step forward in the field of enthesis engineering holding great promise for patients with musculoskeletal injuries and disorders, bridging the gap between laboratory research and real-world clinical applications.