Abstract
Anterior cruciate ligament (ACL) cell sheets combined with biomechanically competent scaffolds might facilitate ACL tissue engineering. Since thermoresponsive polymers allow a rapid enzyme-free detachment of cell sheets, we evaluated the applicability of a thermoresponsive poly(glycidyl ether) (PGE) coating for cruciate ligamentocyte sheet formation and its influence on ligamentocyte phenotype during sheet-mediated colonization of embroidered scaffolds. Ligamentocytes were seeded on surfaces either coated with PGE or without coating. Detached ligamentocyte sheets were cultured separately or wrapped around an embroidered scaffold made of polylactide acid (PLA) and poly(lactic-co-ε-caprolactone) (P(LA-CL)) threads functionalized by gas-phase fluorination and with collagen foam. Ligamentocyte viability, protein and gene expression were determined in sheets detached from surfaces with or without PGE coating, scaffolds seeded with sheets from PGE-coated plates and the respective monolayers. Stable and vital ligamentocyte sheets could be produced within 24 h with both surfaces, but more rapidly with PGE coating. PGE did not affect ligamentocyte phenotype. Scaffolds could be colonized with sheets associated with high cell survival, stable gene expression of ligament-related type I collagen, decorin, tenascin C and Mohawk after 14 d and extracellular matrix (ECM) deposition. PGE coating facilitates ligamentocyte sheet formation, and sheets colonizing the scaffolds displayed a ligament-related phenotype.
Highlights
Cell sheets represent highly confluent monolayers, associated with their self-made extracellular matrix (ECM)
Since thermoresponsive polymers allow a rapid enzyme-free detachment of cell sheets, we evaluated the applicability of a thermoresponsive poly(glycidyl ether) (PGE) coating for cruciate ligamentocyte sheet formation and its influence on ligamentocyte phenotype during sheet-mediated colonization of embroidered scaffolds
Thermoresponsive brushes based on glycidyl methyl ether (GME)/ethyl glycidyl ether (EGE) (1:3) copolymers were self-assembled onto polystyrene 12-well culture plates (Greiner, ThermoFisher Scientific Inc., Darmstadt, Germany) via the physical adsorption of a hydrophobic, photo-reactive benzophenone anchor block based on the monomer 4-[2-(2,3
Summary
Cell sheets represent highly confluent monolayers, associated with their self-made extracellular matrix (ECM). In 1990, the first thermoresponsive coating based on poly(N-isopropylacrylamide) (PNIPAm) was developed on standard tissue culture polystyrene for the production of cell sheets [16,17], which is commercially available under the tradename UpCellTM [18,19] These PNIPAm-coated dishes were applied to harvest sheets of different cell types and have already been used for ligament sheet formation by ACL-derived CD34 positive stem cells [5], ACL fibroblasts and rotator cuff-derived cells [6,20] (Table 1). After basic structure–property relationship studies with PGEs on gold model substrates [27,28], these functional coatings for cell sheet fabrication were transferred to more commonly applied glass [29] and polystyrene cell culture dishes [14,30], which were used in this study to prepare cell sheets with lapine cruciate ligamentocytes These ligamentocyte sheets were seeded on a biomechanically competent embroidered scaffold for ligamentogenesis. ACL: anterior cruciate ligament, ASC: adipose tissue derived stem cells, BMP2: bone morphogenetic protein, CL: cruciate ligament, CTGF: connective tissue growth factor, d: day, h: hour, LSPC: ligament stem/progenitor cells, MSC: mesenchymal stromal cells, PCL: polycaprolactone, PGE: poly(glycidyl ether), SCs: stem cells, TDSC: tendon-derived stem cells, TNMD: tenomodulin, TSPCs: tendon specific progenitor cells, w: weeks
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