Application of a Hyperelastic 3D Printed Scaffold for Mesenchymal Stem Cell-Based Fabrication of a Bizonal Tendon Enthesis-like Construct

Abstract

After surgical tendon repair, the tendon-to-bone enthesis often does not regenerate, which leads to high numbers of rupture recurrences. To remedy this, tissue engineering techniques are being pursued to strengthen the interface and improve regeneration. In this study, we used hyperelastic biphasic 3D printed PLGA scaffolds with aligned pores at the tendon side and random pores at the bone side to mimic the native insertion side. In an attempt to recreate the enthesis, the scaffolds were seeded with adult human mesenchymal stem cells and then cultured in dual fluidic bioreactors, which allows the separate in-flow of tenogenic and chondrogenic differentiation media. MTS assay confirmed the ability of cells to proliferate in dual-flow bioreactors at similar levels to tissue culture plate. Hematoxylin-eosin staining confirmed a compact cell layer entrapped within newly deposited extracellular matrix attached to the scaffolds’ fibers and between the porous cavities, that increased with culture time. After 7, 14, and 21 days, samples were collected and analyzed by qRT-PCR and GAG production. Cultured constructs in dual fluidic bioreactors differentiate regionally toward a tenogenic or chondrogenic fate dependent on exposure to the corresponding differentiation medium. SOX9 gene expression was upregulated (up to 50-fold compared to control) in both compartments, with a more marked upregulation in the cartilaginous portion of the scaffold, By day 21, the cartilage matrix marker, collage II, and the tendon specific marker, tenomodulin, were found to be highly upregulated in the cartilaginous and tendinous portions of the construct, respectively. In addition, GAG production in the treated constructs (serum-free) matched that in control constructs exposed to 10% fetal bovine serum, confirming the support of functional matrix formation in this system. In summary, our findings have validated this dual fluidic system as a potential platform to form the tendon enthesis, and will be optimized in future studies to achieve the fabrication of distinctly biphasic constructs.

Advanced strategies for constructing interfacial tissues of bone and tendon/ligament

“… 110 – 112 However, the development of enthesis TE is not as advanced and the successful reconstruction of enthesis tissue has not yet been achieved. 3 , 26 , 113 – 115 Studies on the use of TE in enthesis repair involve four strategies, the biological scaffold, cell, growth factor, and biophysical modulation strategies. Of these, the biological scaffold strategy has attracted the most interest ( Table 1 ).…”

Advances of Hydrogel-Based Bioprinting for Cartilage Tissue Engineering

“…Poly (L-Lactide-Co-Glycolide) PLGA which consisted of a copolymer of lactide and glycolide had received significant attentions for bio-ink application due to its adjustable structures, good biocompatibility, and biodegradability (Wang et al, 2022). Gottardi et al prepared a biphasic bioprinting PLGA scaffold with embedding adult human mesenchymal stem cells for chondrogenic regeneration (Gottardi et al, 2021). The in vitro assay showed that constructs cultured in a two-fluid bioreactor differentiated locally into tendinocytes or chondrocytes according to exposure to the appropriate differentiation medium after culturing for 7, 14, and 21 days.…”

Generation of Hepatobiliary Cell Lineages from Human Induced Pluripotent Stem Cells: Applications in Disease Modeling and Drug Screening

“…This scaffold promoted hepatocyte survival, hepatic functions and expression of hepatocyte-specific genes [ 103 ]. Thus, not only spatial cell geometry [ 104 ], but also cell differentiation can be controlled by modifying the chemical structure of the hydrogel-based 3D support. This feature is important when handling iPSCs, which can easily undergo undesired phenotypes during in vitro culture.…”