Three-layer collagen-based composite scaffolds to spatially direct tissue-specific cell differentiation for enthesis repair
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Date
2023-09-28Embargo Date
2025-09-25
Author
Pugliese, Eugenia
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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.