Efficient material-induced activation of monocyte-derived dendritic cells releasing surface molecules, matrix metalloproteinases, and growth factors needed for regenerative tissue remodeling.

New experimental approaches for tissue repair have recently been proposed and include the application of natural or synthetic biomaterials and immune cells. Herein, fully synthetic poly(glycidyl ether) (PGE) copolymer coatings are evaluated as bioinstructive materials for the in vitro culture and intrinsic activation of human immune cells. Immature monocyte-derived dendritic cells (moDCs) are exposed to PGE brush and gel coatings of varying copolymer composition, wettability, and deformability immobilized on polystyrene culture dishes. Compared to moDCs cultured on standard tissue culture-treated polystyrene, activation marker levels on the cell surface are strongly enhanced on PGE substrates. Thereby, moDCs undergo a distinct morphological change and reach levels of activation comparable to those achieved by toll-like receptor (TLR) ligand liposaccharide (LPS), specifically for the expression of costimulatory molecules CD86 and CD40 as well as human leukocyte antigen (HLA)-DR. In addition, PGE coatings induce a significantly enhanced level of programmed cell death ligands 1 and 2 (PD-L1/-L2) on the moDC surface, two molecules crucially involved in maintaining immune tolerance. In addition, an increased release of matrix metalloproteinases MMP-1 and MMP-7, as well as transforming growth factor (TGF)-ß1 and epidermal growth factor (EGF) was observed in moDCs cultured on PGE substrates. As fully synthetic biomaterials, PGE coatings demonstrate intrinsic functional competence in instructing immature human moDCs for phenotypic activation in vitro, accompanied by the secretion of bioactive molecules, which are known to be crucial for tissue regeneration. Hence, PGE coatings hold strong potential for immune-modulating implant coatings, while PGE-activated moDCs are promising candidates for future clinical cell-based immunoengineering therapies.

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

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.

Thermoresponsive Poly(glycidyl ether) Brush Coatings on Various Tissue Culture Substrates-How Block Copolymer Design and Substrate Material Govern Self-Assembly and Phase Transition.

Thermoresponsive poly(glycidyl ether) brushes can be grafted to applied tissue culture substrates and used for the fabrication of primary human cell sheets. The self-assembly of such brushes is achieved via the directed physical adsorption and subsequent UV immobilization of block copolymers equipped with a short, photo-reactive benzophenone-based anchor block. Depending on the chemistry and hydrophobicity of the benzophenone anchor, we demonstrate that such block copolymers exhibit distinct thermoresponsive properties and aggregation behaviors in water. Independent on the block copolymer composition, we developed a versatile grafting-to process which allows the fabrication of poly(glycidyl ether) brushes on various tissue culture substrates from dilute aqueous-ethanolic solution. The viability of this process crucially depends on the chemistry and hydrophobicity of, both, benzophenone-based anchor block and substrate material. Utilizing these insights, we were able to manufacture thermoresponsive poly(glycidyl ether) brushes on moderately hydrophobic polystyrene and polycarbonate as well as on rather hydrophilic polyethylene terephthalate and tissue culture-treated polystyrene substrates. We further show that the temperature-dependent switchability of the brush coatings is not only dependent on the cloud point temperature of the block copolymers, but also markedly governed by the hydrophobicity of the surface-bound benzophenone anchor and the subjacent substrate material. Our findings demonstrate that the design of amphiphilic thermoresponsive block copolymers is crucial for their phase transition characteristics in solution and on surfaces.

Endothelial, smooth muscle and fibroblast cell sheet fabrication from self-assembled thermoresponsive poly(glycidyl ether) brushes.

In this study, we introduce a platform to fabricate human dermal fibroblast (HDF), human aortic smooth muscle cell (HAoSMC) and human umbilical vein endothelial cell (HUVEC) sheets using thermoresponsive poly(glycidyl ether) coatings. Copolymer brushes based on glycidyl methyl ether (GME) and ethyl glycidyl ether (EGE) were self-assembled onto polystyrene (PS) culture substrates via the physical adsorption of a hydrophobic, photoreactive benzophenone anchor block based on the monomer 4-[2-(2,3-epoxypropoxy)ethoxy]benzophenone (EEBP). The directed self-assembly of well-defined, end-tethered poly(GME-ran-EGE)-block-poly(EEBP) (PGE) brushes was achieved via the selective, EEBP-driven adsorption of the asymmetric block copolymer from dilute aqueous solution below its cloud point temperature (CPT). Subsequently, the PGE brush layers were covalently immobilized onto the PS surfaces by irradiation with UV light and characterized by ellipsometry, static water contact angle (CA) measurements and atomic force microscopy (AFM). We found that, by decreasing the temperature from 37 to 20 °C, the coatings undergo a pancake-to-brush transition, which triggers cell sheet detachment. In addition, cell culture parameters were optimized to allow proper adhesion and controlled detachment of confluent HDF, HAoSMC and HUVEC sheets, which can be applied in vascular tissue engineering.

Switchable Oligo(glycidyl ether) Acrylate Bottlebrushes "Grafted-from" Polystyrene Surfaces: A Versatile Strategy toward Functional Cell Culture Substrates.

Thermoresponsive brushes based on linear poly(glycidyl ether)s (PGEs) have already shown to be functional coatings for cell sheet fabrication. In here, we introduce a method to functionalize polystyrene (PS) tissue culture substrates with thermoresponsive coatings comprising glycidyl ether-based bottlebrush architectures. Utilizing the UV-induced "grafting-from" approach, thermoresponsive oligo(glycidyl ether) acrylate (OGEA) macromonomers were polymerized from PS substrates under bulk conditions. Applying ellipsometry, water contact angle (CA), and atomic force microscopy (AFM) measurements, we found that OGEA coatings exhibit a complex, gel-like structure comprising nanosized roughness and exhibit a temperature-dependent phase transition in water through the reversible hydration of OGEA bottlebrush side chains. To assess the utility of the coatings as functional substrates for cell sheet fabrication, human dermal fibroblast (HDF) adhesion and detachment were investigated. By adjusting the bottlebrush properties via the grafting procedure and coating structure, we were able to harvest confluent HDF sheets from functionalized PS substrates in a temperature-triggered, controlled manner. As the first report on surface-grafted bottlebrushes comprising thermoresponsive side chains with molecular weight of up to 1 kDa, this study demonstrates the potential of OGEA-based coatings for cell sheet fabrication.

Dynamic Protein Adsorption onto Dendritic Polyglycerol Sulfate Self-Assembled Monolayers.

Biomaterial surfaces that are in contact with blood are often prone to unspecific protein adsorption and the activation of the blood clotting cascade. Hence, such materials usually must be functionalized with low-fouling or anticoagulant polymer coatings to increase their performance and durability with respect to various applications, for example as implants or in biomedical devices. Many coatings are based on anionic polymers, such as heparin, and are known to have pronounced anticoagulant effects. To assess the ability of a surface to prevent biofouling and to get further insight into its underlying mechanism, studies of the protein adsorption on self-assembled monolayers (SAMs) are often used as a predictive tool. In this article, we synthesized thioctic acid-functionalized dendritic polyglycerol sulfate (dPGS), which is a well-known synthetic heparin mimetic, and immobilized it onto gold model surfaces. The anionic dPGS SAMs were characterized via contact angle measurements and ellipsometry and compared to their neutral dendritic polyglycerol (dPG) counterparts with respect to their single protein adsorption of the two most abundant blood proteins albumin (Alb) and fibrinogen (Fib). In addition, we used QCM-D and ToF-SIMS as complementary techniques to investigate the dynamic, mixed, and sequential adsorption of Alb and Fib. Our results clearly demonstrate an incomplete Vroman effect and indicate the rearrangement of the adsorbed protein layers, which is presumably drive by ionic interactions between the two proteins and the anionic dPGS surface.

Ultrathin Poly(glycidyl ether) Coatings on Polystyrene for Temperature-Triggered Human Dermal Fibroblast Sheet Fabrication.

The fabrication of cell sheets is a major requirement for bottom-up tissue engineering purposes (e.g., cell sheet engineering) and regenerative medicine. Employing thermoresponsive polymer coatings as tissue culture substrates allows for the mild, temperature-triggered detachment of intact cell sheets along with their extracellular matrix (ECM). It has been shown before that biocompatible, thermoresponsive poly(glycidyl ether) monolayers on gold substrates can be utilized to harvest confluent cell sheets by simply reducing the temperature to 20 °C. Herein, we report on the first poly(glycidyl ether)-based coating on an application-relevant tissue culture plastic substrate. We devised a simple, substrate-geometry-independent method to functionalize polystyrene (PS) surfaces from dilute ethanolic solution via the physical adsorption process of a thermoresponsive poly(glycidyl ether) block copolymer (PGE) bearing a short, hydrophobic, and photoreactive benzophenone (BP) anchor block. Subsequently, the PGE-coated PS is UV-irradiated for covalent photoimmobilization of the polymer on the PS substrate. Online monitoring of the adsorption process via QCM-D measurements and detailed characterization of the resulting coatings via AFM, ellipsometry, and water contact angle (CA) measurements revealed the formation of an ultrathin PGE layer with an average dry thickness of 0.7 ± 0.1 nm. Adhesion and proliferation of human dermal fibroblasts on PGE-coated PS and tissue culture PS (TCPS) were comparable. For temperature-triggered detachment, fibroblasts were cultured in PGE-coated PS culture dishes at 37 °C for 24 h until they reached confluency. Intact cell sheets could be harvested from the thermoresponsive substrates within 51 ± 17 min upon cooling to 20 °C, whereas sheets could not be harvested from uncoated PS and TCPS control dishes. Live/dead staining and flow cytometry affirmed a high viability of the fibroblasts within the cell sheets. Hence, ultrathin layers of thermoresponsive poly(glycidyl ether)s on hydrophobic PS substrates are functional coatings for cell sheet fabrication.