Three-layer collagen-based composite scaffolds to spatially direct tissue-specific cell differentiation for enthesis repair
Pugliese, Eugenia
Pugliese, Eugenia
Loading...
Repository DOI
Publication Date
2023-09-28
Type
Thesis
Downloads
Citation
Abstract
The enthesis is a specialised interfacial tissue responsible to minimise stress concentrations between tendon and bone. Fibrocartilaginous entheses are composed of four distinct areas, namely tendon, unmineralised fibrocartilage, mineralised fibrocartilage and bone, and they are characterised by spatial gradients of cell phenotype, matrix composition and organisation. Given the intrinsic complexity of this tissue and the inherently poor healing of tendons and ligaments, regeneration of the enthesis is particularly challenging, usually resolving in a scar populated by fibroblasts. Surgical approaches still fail to restore the native fibrocartilaginous transition, while tissue engineering strategies have offered some valid approaches. Among the most promising strategies, multiphasic scaffolds seeded with adult differentiated cell types or adult mesenchymal stromal cells are worth of mentioning. Although the former strategy exploits the heterotypic interactions between different cell phenotypes in proximity to each other, it poses considerable scalability and regulatory challenges. The latter approach benefits from the use of a single cell population (making its applicability far easier) that will differentiate towards the appropriate tissue lineage through scaffold- and local microenvironment- induced signals. Recent advances in biomaterial engineering have inspired the development of multi-cargo delivery vehicles to mimic naturally occurring gradients in composition, signalling cues and other constituents of the enthesis but, despite the very promising preclinical data, none of them has been clinically translated yet. Herein, the potential of a three-layer enthesis-composition inspired collagen - based scaffold to spatially direct differentiation of human bone marrow derived mesenchymal stromal cells in an in vitro model or maintain phenotype of native tendon-derived cells in an ex vivo model, with the aid of a zonal functionalisation with bioactive molecules were assessed. Before approaching the design of a multi-layer collagen - based scaffold, hydrogels and sponges were singularly tested as monolayer scaffolds and their production and crosslinking were optimised. Collagen type I hydrogels were fabricated with in-house extracted porcine collagen and cross-linked with polyethylene glycol succinimidyl succinate molecule, tested at different number of arms (4 and 8), molecular weights (10, 20 and 40 KDa) and concentrations (0, 0.1, 0.5, 1, 2.5 and 5 mM). Although some of the conditions yielded stable hydrogels, they degraded too quickly in cell culture conditions, so they were deemed unsuitable for in vitro studies. The focus was shifted towards more clinically relevant scaffolds for enthesis repair, such as sponges, and the cross-linker of choice was changed to 4-arm 10 KDa polyethylene glycol succinimidyl glutarate. Collagen type I and collagen type II monolayer sponges were produced and their crosslinking density optimised. Afterwards, the 3-L scaffold production was optimised by testing different freezing and freeze-drying process to finally obtain an organised porous network by iterative layering freeze-drying. This process yielded three interconnected, yet distinguishable layers that mimicked the basic extracellular matrix composition of the enthesis: a bone-like layer made of collagen type I and hydroxyapatite, a fibrocartilage-like layer made of collagen type II and a tendon-like layer made of collagen type I. To assess the potential of the scaffold to promote specific cell lineage commitment, human bone marrow derived mesenchymal stromal cells were seeded onto the scaffolds and their differentiation towards the three cell populations of the enthesis tissue was investigated. Cells infiltrated and homogeneously spread throughout the scaffold; as a response to the composition of the scaffold, cells differentiated in a localised manner in the bone-like layer towards the osteogenic lineage and, in combination with differentiation medium, towards the fibrocartilage lineage. To aid tenogenic and fibrochondrogenic differentiation, different bioactive molecules were screened in supplementation to basal medium on cell - seeded tendon-like layer and fibrocartilage-like layer monolayer scaffolds. The best two candidates [for tenogenic differentiation: platelet-derived growth factor bb and transforming growth factor β3; for fibrochondrogenic differentiation: transforming growth factor β3 and bone morphogenetic protein 2] were incorporated in the tendon-like layer or the fibrocartilage-like layer during the fabrication process and their effect was assessed. To better simulate an in vivo implantation scenario, the three-layer scaffolds were placed in proximity of Achilles rat tendons to assess native tendon-derived cell response. In the absence of bioactive molecule functionalisation, the scaffolds were fully populated and a fibrocartilage interface was initiated, as evidenced by collagen type II presence in the fibrocartilage-like layer. Overall, these results indicate that the three-layer composite collagen scaffolds can stimulate osteogenic and fibrochondrogenic differentiations, even in the absence of bioactive molecule functionalisation, which can help to strengthen the attachment between bone and repaired tendon and lay the foundations for an in vivo functional repair of the enthesis.
Funder
Publisher
NUI Galway