Development of three-layer collagen scaffolds to spatially direct tissue-specific cell differentiation for enthesis repair

Enthesis repair remains a challenging clinical indication. Herein, a three-layer scaffold composed of a tendon-like layer of collagen type I, a fibrocartilage-like layer of collagen type II and a bone-like layer of collagen type I and hydroxyapatite, was designed to recapitulate the matrix composition of the enthesis. To aid tenogenic and fibrochondrogenic differentiation, bioactive molecules were loaded in the tendon-like layer or the fibrocartilage-like layer and their effect was assessed in in vitro setting using human bone marrow derived mesenchymal stromal cells and in an ex vivo model. Seeded human bone marrow mesenchymal stromal cells infiltrated and homogeneously spread throughout the scaffold. As a response to the composition of the scaffold, cells differentiated in a localised manner towards the osteogenic lineage and, in combination with differentiation medium, towards the fibrocartilage lineage. Whilst functionalisation of the tendon-like layer did not improve tenogenic cell commitment within the time frame of this work, relevant fibrochondrogenic markers were detected in the fibrocartilage-like layer when scaffolds were functionalised with bone morphogenetic protein 2 or non-functionalised at all, in vitro and ex vivo, respectively. Altogether, our data advocate the use of compartmentalised scaffolds for the repair and regeneration of interfacial tissues, such as enthesis.