Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies

Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex-vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 h in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitro

Macromolecular crowding meets tissue engineering by self-assembly

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\u27 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.2016-05-1

La parentalité en Afrique de l’Ouest et du Centre

International audienceLa « perspective genre » s'impose aujourd'hui comme une grille d'analyse incontournable dans les recherches en sciences sociales et dans la recherche africaniste notamment. Cette avancée doit beaucoup aux études sur les femmes qui se sont multipliées au cours des dernières décennies en lien avec le vaste champ de réflexion que représente le développement. Prendre en compte l'importance des rapports sociaux de sexe pour mieux comprendre le fonctionnement des sociétés et pour apporter des réponses aux populations est certes admis en théorie mais dans les faits loin d'être un réflexe pour tous. Cette démarche, trop souvent assimilée à une posture militante, peine à trouver sa légitimité sur la scène politique et scientifique, en Afrique tout particulièrement. Les femmes intellectuelles africaines qui dès les années 1975 se sont mobilisées, ont récusé avec ferveur le fait que le discours sur « les Africaines comme objets de l'anthropologie » soit complètement accaparé par les féministes occidentales. Elles étaient alors animées par la nécessité de développer les conditions de production d'un discours sur elles-mêmes produit par elles-mêmes. Parallèlement « les Africaines ne cessaient de dire qu'elles vivaient dans des contextes différents et que leurs objectifs n'étaient pas forcément les mêmes » (Sow, 2008 : 13) que ceux poursuivis par les tenantes des women's studies anglo-saxonnes. Aujourd'hui, nombre d'intellectuelles africaines se mobilisent dans les universités du continent pour promouvoir des recherches autour du genre et des nouveaux enjeux contemporains qui appellent une lecture sous l'angle du genre pour en saisir toute la complexité

Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies

Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 h in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitroHealth Research BoardScience Foundation IrelandCollege of Engineering & Informatics, National University of Ireland, Galway, Postgraduate College Fellowshi

Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies.

Journal articleTherapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 h in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitro.Science Foundation Ireland, Research Frontiers Programme, Project Number: SFI-09-RFP-ENM248

Macromolecular crowding meets tissue engineering by self-ssembly: A paradigm shift in regenerative medicine

Advancements in molecular and cell biology have led to the development of cell-based therapies to treat injured or degenerated tissues. [1] The rationale of this concept is that functional regeneration can be achieved best by using the innate capacity of cells to create their own tissue-specific extracellular matrix (ECM) avoiding the shortfalls of man-made devices. Although direct cell injections have demonstrated very promising preclinical and clinical outcomes, [2] the mode of administration offers little control over local retention and distribution of the injected cell suspensions[3] leading to scattered therapeutic efficiency. This deficiency has led to the development of living substitutes for skin[4] and blood vessel[5] composed of cells seeded on a collagen scaffold. Notwithstanding the efficacious results in preclinical models and clinical trials, it soon became apparent that the presence of the scaffold hinders tissue remodelling and function. [6] These drawbacks led to the development of the scaffold-free cell-sheet tissue engineering (CSTE)[7] or tissue engineering by self-assembly (TESA), [8] a therapy that offers the fabrication of a contiguous cell sheet that is stabilised by cell-cell contacts and endogenously produced ECM. Despite the documented, in preclinical and clinical setting, positive outcomes for skin, [9] blood vessel, [10, 11] cornea, [12, 13] heart, [14] lung, [15] liver[16] and bone[17] replacement, only Epicel® (Genzyme, USA) for skin and LifeLine™ for blood vessel (Cytograft, USA) have been commercialised so far. This limited technology transfer from bench-top to clinic has been attributed to the substantial long period of time required for ex vivo culture (e.g. 14-35 days for corneal epithelium; [13] 84 days for corneal stromal; [18] 28 days for corneal endothelium;[19] 70 days for lung cell-sheet; [15] and 196 days for blood vessel[11] ) that often leads to loss of native phenotype and cell senescence.[20] Here, we propose a biophysical approach, termed macromolecular crowding (MMC), that increases thermodynamic activities and biological processes by several orders of magnitude,[21] as means to create ECM-rich tissue equivalents. The principle of MMC is derived from the notion that in vivo cells reside in a highly crowded/dense extracellular space and therefore the conversion of the de novo synthesised procollagen to collagen I is rapid. [22] However, in the even substantially more dilute than body fluids (e.g. urine: 36-50g/l; blood: 80g/l) culture conditions (e.g. HAM F10 nutrient medium: 16.55g/l; DMEM/F12 medium: 16.78g/l; DMEM high glucose and L-glutamine medium: 17.22g/l), the rate limiting conversion of procollagen to collagen I is very slow (Figure 1a). We propose that the addition of inert polydispersed macromolecules (presented as spherical objects of variable diameter in Figure 1b) in the culture media will facilitate amplified production of ECM-rich living substitutes.We thank Dr Oliver Carroll for laboratory management; and Mr Maciek Doczyk (http://doczykdesign.com) for his support in the preparation of Figure 1 of this manuscript. This work is supported by Science Foundation Ireland, Research Frontiers Programme, Project Number: SFI‐09‐RFP‐ENM2483 to D.Z.; Science Foundation Ireland, E.T.S. Walton Visitor Awards Programme, Project Number: 08/W.1/B2568 to M.R., A.P. and D.Z.; Health Research Board, Project Number: HRA_POR/2011/84 to D.Z.; and College of Engineering and Informatics, Postgraduate Scholarship Scheme, NUI Galway to P.K. and D.Z.peer-reviewe

C3a Enhances the Formation of Intestinal Organoids through C3aR1

C3a is important in the regulation of the immune response as well as in the development of organ inflammation and injury. Furthermore, C3a contributes to liver regeneration but its role in intestinal stem cell function has not been studied. We hypothesized that C3a is important for intestinal repair and regeneration. Intestinal organoid formation, a measure of stem cell capacity, was significantly limited in C3-deficient and C3a receptor (C3aR) 1-deficient mice while C3a promoted the growth of organoids from normal mice by supporting Wnt-signaling but not from C3aR1-deficient mice. Similarly, the presence of C3a in media enhanced the expression of the intestinal stem cell marker leucine-rich repeat G-protein-coupled receptor 5 (Lgr5) and of the cell proliferation marker Ki67 in organoids formed from C3-deficient but not from C3aR1-deficient mice. Using Lgr5.egfp mice we showed significant expression of C3 in Lgr5+ intestinal stem cells whereas C3aR1 was expressed on the surface of various intestinal cells. C3 and C3aR1 expression was induced in intestinal crypts in response to ischemia/reperfusion injury. Finally, C3aR1-deficient mice displayed ischemia/reperfusion injury comparable to control mice. These data suggest that C3a through interaction with C3aR1 enhances stem cell expansion and organoid formation and as such may have a role in intestinal regeneration