Macromolecular crowding meets tissue engineering by self-assembly
MetadataShow full item record
This item's downloads: 677 (view details)
Introduction: Advancements in molecular and cell biology have led to the development of tissue engineering by self-assembly. The driving hypothesis of this concept is that replacement, repair and restoration of lost tissue function can be accomplished best by using the cells' inherent capacity to create highly sophisticated structures with precision and efficiency still unmatched by human-made devices. However, the prolonged culture time required to develop an implantable device jeopardises clinical translation and commercialisation. It has been demonstrated that macromolecular crowding enhances the deposition of extracellular matrix. Herein, the influence of crowding molecules on matrix deposition and the potential of this technology in tissue engineering by self-assembly was investigated. Materials and Methods: Human fibroblasts (lung and skin), tenocytes and osteoblasts were cultured under various MMC conditions (dextran sulphate, Ficoll® & carrageenan) in a range of fetal bovine serum (FBS) and human serum (HS) concentrations (0.0-10%). ECM deposition was verified by SDS-PAGE, immunocytochemistry (ICC), atomic force microscopy (AFM), scanning electron microscopy (SEM) and mass-spectrometry (MS). The MMC molecules were characterized by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). The influence of crowders on cell morphology, cell viability and metabolic activity were evaluated using phase-contrast microscopy, Live/Dead® and AlamarBlue® assays respectively. pNIPAAm and pNTBA based thermo-responsive copolymers were developed to facilitate detachment of ECM-rich cell-sheets. Results: The SDS-PAGE and densitometry demonstrated that MMC significantly increases the collagen type-I deposition (p<0.0001) at all tested serum concentrations (maximum deposition was in 2 days & 0.5% FBS or HS). ICC, AFM and SEM further confirmed enhanced deposition of fibrillar ECM in presence of MMC. DLS and NTA demonstrated that CR has highest polydispersity among all tested crowders. Phase-contrast microscopy, Live/Dead® and AlamarBlue® assays confirmed that cellular morphology, viability and metabolic activity respectively were not affected by MMC. Thermo-responsive coating with 65% pNIPAAm: 35% pNTBA facilitated detachment of ECM rich cell-sheet from culture. Complementary ICC for MS validation confirmed the enhanced deposition of collagens (III, IV, V, VI) and other ECM molecules (laminin, fibronectin, hyaluronic acid, decorin, lysyl oxidase), without changing collagen-VII, elastin, fibrillin-1, transglutaminase-2, alpha-smooth muscle actin, epithelial keratin, tubulin, chondroitin sulphate, keratin sulphate, heparin sulphate, aggrecan, biglycan, CD248 and IL-10. Discussion and conclusions: This work reports that the efficacy of macromolecular crowding in enhancing matrix deposition is amplified in human fibroblast, tenocyte and osteoblast cultures in the presence of low serum concentration, due to the low proteolytic activity of serum; in fact, an over 80-fold increase in extracellular matrix deposition is documented within 48 hours. It further identifies that macromolecular polydispersity is key modulator of extracellular matrix deposition, due to the generation of effective volume exclusion effect. Using a custom-made thermal responsive polymer, living substitutes with tissue-specific protein composition and structure were attained. This approach enables modulation of the in vitro microenvironment, without negatively impacting on cellular functions, and therefore opens new avenues for a more rational design in engineering cohesive tissue modules.
This item is available under the Attribution-NonCommercial-NoDerivs 3.0 Ireland. No item may be reproduced for commercial purposes. Please refer to the publisher's URL where this is made available, or to notes contained in the item itself. Other terms may apply.
The following license files are associated with this item: