Cruciate Ligament Cell Sheets Can Be Rapidly Produced on Thermoresponsive poly(glycidyl ether) Coating and Successfully Used for Colonization of Embroidered Scaffolds

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.

Minispheroids as a Tool for Ligament Tissue Engineering: Do the Self-Assembly Techniques and Spheroid Dimensions Influence the Cruciate Ligamentocyte Phenotype?

“…However, in spheroids manufactured with 1000 cells by both techniques, there was a lower gene expression for COL1A1 detectable when compared to monolayer cultures. This was previously observed comparing another type of 3D culture, namely cell sheets, with monolayers using exactly the same cell type [32]. It could be explained by the fact that monolayer cells were deprived of their freshly produced ECM, which is rich in collagen type 1 during passaging, leading to a stimulation of the de novo collagen type 1 synthesis.…”

“…In previous work, we could show that cruciate ligamentocyte spheroids are suitable for the colonization of synthetic polymer scaffolds [18,19,24]. When spheroids [4,22] or cell sheets [32] are seeded onto the scaffolds, cells emigrate and spread onto the scaffolds. However, high numbers of spheroids are required for tissue engineering; hence, high-throughput strategies for spheroid formation are needed.…”

Polyethers Based on Short-Chain Alkyl Glycidyl Ethers: Thermoresponsive and Highly Biocompatible Materials

“…The polymerizations of GME and EGE were initiated by a dibenzyl-protected amino alkyl alkoxide, which allows for the synthesis of α,ω-telechelic polyethers. , After the polymerization, the benzyl protective groups of the initiator were removed by hydrogenation (H 2 , Pd/C), yielding an amine in α-position of the polymer chain, permitting subsequent reactions, which will be discussed below. Schmalz and co-workers as well as Haag, Weinhart, and co-workers, extensively exploited the copolymerization of GME and EGE by AROP to control aqueous solubility and the corresponding LCST. − Copolymers of varying comonomer ratios with M̅ n of up to 4.9 kg mol –1 and narrow dispersities were successfully synthesized.…”

“…Surface-attached brushes of these copolymers on PS culture substrates were employed for cell growth and the fabrication of human dermal fibroblast cells (HDF), human aortic smooth muscle cells (HAoSMC), and human umbilical vein endothelial cell (HUVEC) sheets . The copolymers provide excellent biocompatibility and inhibition of nonspecific protein interaction in analogy to PEG, but additionally offer thermal switchability, e.g., enabling thermally triggered cell detachment in tissue cultures (Figure c). , Recently, Schulze-Tanzil and co-workers used this knowledge for fabrication of anterior cruciate ligament cell (ACL) sheets, which were utilized for colonization of embroidered scaffolds . Herein, the thermoresponsive P­(GME- co -EGE) copolymers enable the thermal, enzyme-free detachment of cell sheets.…”

“…43,86 Recently, Schulze-Tanzil and co-workers used this knowledge for fabrication of anterior cruciate ligament cell (ACL) sheets, which were utilized for colonization of embroidered scaffolds. 48 Herein, the thermoresponsive P(GME-co-EGE) copolymers enable the thermal, enzyme-free detachment of cell sheets. The utilization of thermoresponsive polymers like, e.g., P(GME-co-EGE) for the modulation of the protein corona and materials−cell interaction is outlined in-depth by a recent review of Landfester and co-workers.…”