Tendon lesions are one of the most relevant musculoskeletal sources of injuries worldwide. Among them, the most frequent site of rupture is located at the enthesis (i.e. tendon-to-bone junction). This is caused by complex collagen fibrils’ structure and mechanics of this interfacial tissue region. In fact, the enthesis, designed by nature to connect soft (tendon) and hard (bone) tissues, is mainly composed of four distinct but continuous extracellular matrix (ECM) regions populated by different cells: tendon (aligned collagen fibrils; tenocytes), non-mineralized fibrocartilage (progressive loss of fibrils alignment and conical shape; fibrochondrocytes), mineralized fibrocartilage (increment of random orientation of fibrils; hypertrophic fibrochondrocytes) and bone (fibrils organized in trabecular bone tissue; osteocytes). Moreover, starting from the mineralized fibrocartilage, ECM assumes a progressive gradient of mineralization/stiffness approaching the bone. To avoid abrupt ruptures at the enthesis, the non-mineralized fibrocartilage assumes a typical conical structure, able to self-expand in response to a load (i.e. auxetic behavior), serving as a stress concentration reducer.
Due to the morphological/mechanical similarities with the human tissue, large animal models, especially the sheep ones, are often used to validate innovative implantable regenerative or prosthetic devices in vivo.
From a regenerative perspective, dedicated biofabrication strategies are often applied to regenerate the different regions of the enthesis. In particular, electrospinning (ES), and its polymeric nanofibers, is the most suitable technique to mimic the collagen fibril structure of tendons and fibrocartilage, while additive manufacturing (AM) is the perfect strategy to reproduce the bone tissue. These scaffolds have widely demonstrated to enhance stem cell proliferation and differentiation when these cells are cultured on them.
However, despite the preliminarily results shown in literature, a method to faithfully mimic the target tendon-enthesis-bone chain of interest, driving the stem cells fate, is unexplored so far.
The aim of 3NTHESES was to fill these scientific gaps by developing the first multi-layered hierarchical multi-material scaffold (MLHMMS) able to biomimetically replicate the multiscale structure/mechanics of a target sheep tendon-enthesis-bone complex of interest. This ambitious goal was achieved by combining the skills in the unique hierarchical ES technique developed by the applicant (Dr. Alberto Sensini) to mimic the tendon tissue and his skills in morphological/mechanical characterizations of biological/biofabricated tissues, with the top-notch AM, stem cells and enthesis knowledge of the host institution (Maastricht University, Profs. Lorenzo Moroni and Martijn van Griensven).
Specifically, the objectives of 3NTHESES were:
1. To study the morphology and mechanics of a relevant target animal model of interest (i.e. sheep calcaneal tendon) by using a mix of imaging techniques, biological evaluations and imaging-based mechanical characterizations. This to parallelly acquire data to replicate the tissue and increase the limited biomechanical knowledge concerning this relevant animal model.
2. To develop and characterize a panel of innovative ES scaffolds to mimic, with a bottom-up hierarchical approach, the whole target sheep tendon and enthesis tissues.
3. To develop procedures to match image-based biomimetic AM bone scaffolds and ES tendon-enthesis biomimetic scaffolds to produce innovative MLHMMS able to drive the stem cells fate.
3NTHESES reached all the objectives opening the way for new research to maximize its scientific impact.
