Abstract
Defining the best combination of cells and biomaterials is a key challenge for the development of tendon tissue engineering (TE) strategies. Adipose-derived stem cells (ASCs) are ideal candidates for this purpose. In addition, controlled cell-based products adherent to good manufacturing practice (GMP) are required for their clinical scale-up. With this aim, in this study, ASC 3D bioprinting and GMP-compliant tenogenic differentiation were investigated. In detail, primary human ASCs were embedded within a nanofibrillar-cellulose/alginate bioink and 3D-bioprinted into multi-layered square-grid matrices. Bioink viscoelastic properties and scaffold ultrastructural morphology were analyzed by rheology and scanning electron microscopy (SEM). The optimal cell concentration for printing among 3, 6 and 9 × 106 ASC/mL was evaluated in terms of cell viability. ASC morphology was characterized by SEM and F-actin immunostaining. Tenogenic differentiation ability was then evaluated in terms of cell viability, morphology and expression of scleraxis and collagen type III by biochemical induction using BMP-12, TGF-β3, CTGF and ascorbic acid supplementation (TENO). Pro-inflammatory cytokine release was also assessed. Bioprinted ASCs showed high viability and survival and exhibited a tenocyte-like phenotype after biochemical induction, with no inflammatory response to the bioink. In conclusion, we report a first proof of concept for the clinical scale-up of ASC 3D bioprinting for tendon TE.
Highlights
The inability of native tendons to de novo synthesize their extracellular matrix (ECM) and restore their functions after injury is expected to be overcome by cell-based tissue engineering
In order to move towards the clinical application of a safe and standardized Adipose-derived stem cells (ASCs)-based product adherent to good manufacturing practice (GMP), clinical-grade reagents were selected as TENO medium constituents, avoiding the use of products derived from animal sources [18]
We demonstrated that ASCs cultured in TENO xenogenic-free medium exhibited a tenocyte-like phenotype by expression of specific tendon-related markers such as scleraxis, collagen type I/III, tenomodulin, COMP and metalloproteinase-1/-3
Summary
The inability of native tendons to de novo synthesize their extracellular matrix (ECM) and restore their functions after injury is expected to be overcome by cell-based tissue engineering. The 3D environment plays a key role in driving cell behavior and fate further and scaffolding strategies could be exploited to design suitable cell substrates for tendon TE mimicking the specific ECM composition and the multiple hierarchical collagen fibrous structure of tendon tissue [19]. In this context, nanofibrous materials have recently been studied because of their natural microporous structure resembling the ultrastructure of tendon tissue, that could provide cell–cell interactions and proper porosity to encourage remodeling [20].
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