Physico-chemical characterization of Antheraea mylitta silk mats for wound healing applications.

In the field of plastic reconstructive surgery, development of new innovative matrices for skin repair is in demand. The ideal biomaterial should promote attachment, proliferation and growth of cells. Additionally, it should degrade in an appropriate time period without releasing harmful substances, not exerting a pathological immune response. The materials used should display optimized mechanical properties to sustain cell growth and limit scaffold contraction. Wound healing is a biological process directed towards restoration of tissue that has suffered an injury. An important phase of wound healing is the generation of a basal epithelium wholly replacing the epidermis of the wound. Wild silk from Antheraea mylitta meets these demands to a large extent. To evaluate the effects of the treatment, Antheraea mylitta and Bombyx mori samples were characterized by SEM-EDX, FT-IR, XRD and TGA-DSC techniques. Preliminary cell growth behavior was carried out by culturing epidermal cells and proliferation was quantified via viability assay. Moreover, Antheraea mylitta possesses excellent cell-adhesive capability, effectively promoting cell attachment and proliferation. Antheraea mylitta serves as a delivery vehicle for cells. With all these unique features, it is expected that Antheraea mylitta mat will have wide utility in the areas of tissue engineering and regenerative medicine.Prof. Shyamkumar Vootla and Dr. Julien Gautrot thanks to Department of Science and Technology (DST), New Delhi. India and United Kingdom India Education and Research Initiative (UKIERI), British Council, United Kingdom, for funding of this project (DST/INT/UK/P-52). Tis work was supported by Commonwealth Academic Fellowship awarded to Prof. Shyamkumar Vootla by the Commonwealth Association of Universities, United Kingdom

Biofunctionalized Patterned Polymer Brushes via Thiol-Ene Coupling for the Control of Cell Adhesion and the Formation of Cell Arrays

Thiol–ene radical coupling is increasingly used for the biofunctionalization of biomaterials. Thiol–ene chemistry presents interesting features that are particularly attractive for platforms requiring specific reactions with peptides or proteins and the patterning of cells, such as reactivity in physiological conditions and photoactivation. In this work, we synthesized alkene-functionalized (allyl and norbornene residues) antifouling polymer brushes (based on poly­(oligoethylene glycol methacrylate)) and studied thiol–ene coupling with a series of thiols including cell adhesive peptides RGD and REDV. The adhesion of umbilical vein endothelial cells (HUVECs) to these interfaces was studied and highlighted the absence of specific integrin engagement to REDV, in contrast to the high level of cell spreading observed on RGD-functionalized polymer brushes. This revealed that α₄β₁ integrins (binding to REDV sequences) are not sufficient on their own to sustain HUVEC spreading, in contrast to α_vβ₃ and α₅β₁ integrins. In addition, we photopatterned peptides at the surface of poly­(oligoethylene glycol methacrylate) (POEGMA) brushes and characterized the quality of the resulting arrays by epifluorescence microscopy and atomic force microscopy (AFM). This allowed the formation of cell patterns and demonstrated the potential of thiol–ene based photopatterning for the design of cell microarrays

Photoconfigurable, Cell-Remodelable Disulfide Cross-linked Hyaluronic Acid Hydrogels.

Dynamic photoresponsive synthetic hydrogels offer important advantages for biomaterials design, from the ability to cure hydrogels and encapsulate cells in situ to the light-mediated control of cell-spreading and tissue formation. We report the facile and effective photocuring and photoremodeling of disulfide-cross-linked hyaluronic acid hydrogels, based on photo-oxidation of corresponding thiol residues and their radical-mediated photodegradation. We find that the mechanical properties of disulfide hydrogels and the extent of their photoremodeling can be tuned by controlling the photo-oxidation and photodegradation reactions, respectively. This enables not only the photopatterning of the mechanical properties of hydrogels but also their self-healing and photomediated healing. Finally, we demonstrate the ability to encapsulate mesenchymal stromal cells within these materials and to regulate their protrusion and spreading in 3D matrices by controlling the mechanical properties of the disulfide networks. Therefore, synthetically accessible photoconfigurable disulfide hydrogels offer interesting opportunities for the design of soft biomaterials and the regulation of cell encapsulation and matrix remodeling for tissue engineering

Contractile myosin rings and cofilin-mediated actin disassembly orchestrate ECM nanotopography sensing

The nanotopography and nanoscale geometry of the extra-cellular matrix (ECM) are important regulators of cell adhesion, motility and fate decision. However, unlike the sensing of matrix mechanics and ECM density, the molecular processes regulating the direct sensing of the ECM nanotopography and nanoscale geometry are not well understood. Here, we use nanotopographical patterns generated via electrospun nanofibre lithography (ENL) to investigate the mechanisms of nanotopography sensing by cells. We observe the dysregulation of actin dynamics, resulting in the surprising formation of actin foci. This alteration of actin organisation is regulated by myosin contractility but independent of adapter proteins such as vinculin. This process is highly dependent on differential integrin expression as β3 integrin expressing cells, more sensitive to nanopattern dimensions than β1 integrin expressing cells, also display increased perturbation of actin assembly and actin foci formation. We propose that, in β3 integrin expressing cells, contractility results in the destabilisation of nanopatterned actin networks, collapsing into foci and sequestering regulators of actin dynamics such as cofilin that orchestrate disassembly. Therefore, in contrast to the sensing of substrate mechanics and ECM ligand density, which are directly orchestrated by focal adhesion assembly, we propose that nanotopography sensing is regulated by a long-range sensing mechanism, remote from focal adhesions and mediated by the actin architecture

Surface-Initiated Poly(oligo(2-alkyl-2-oxazoline)methacrylate) Brushes

Polymer brushes are particularly performant antifouling coatings, owing to their high grafting density that prevents unwanted biomacromolecules to diffuse through the coating and adhere to the underlying substrate. In addition to this structural feature, polymer brushes require a relatively high level of hydrophilicity and a globally neutral structure to display ultrahigh protein resistance. Poly­(2-alkyl-2-oxaolines) are attractive building blocks for such coatings as they can display relatively high hydrophilicity, owing to their amide repeat units, but can also be side-chain and end-chain functionalized relatively readily. However, poly­(2-alkyl-2-oxazolines) have not yet been introduced through a radical-mediated grafting from polymer brush structure that would confer the high level of grafting density that is the hallmark of highly protein resistant brushes. Here, we present the formation of a series of poly­(oligo­(2-alkyl-2-oxazoline)­methacrylate) brushes generated via a grafting from approach, via atom transfer radical polymerization. We characterize the chemical structure of the resulting coatings via ellipsometry, Fourier-transform infrared spectroscopy, and X-ray photoelectron spectroscopy. We show that allyl end groups can be introduced as a side chain of these brushes to allow functionalization via thiol-ene chemistry. We demonstrate the excellent protein resistance of these coatings in single protein solutions as well as serum solutions at concentration typically used for cell culture. Finally, we demonstrate the feasibility of using these brushes for the micropatterning of cells and the generation of cell-based assays

Vascularised cardiac spheroids-on-a-chip for testing the toxicity of therapeutics.

Microfabricated organ-on-a-chips are rapidly becoming the gold standard for the testing of safety and efficacy of therapeutics. A broad range of designs has emerged, but recreating microvascularised tissue models remains difficult in many cases. This is particularly relevant to mimic the systemic delivery of therapeutics, to capture the complex multi-step processes associated with trans-endothelial transport or diffusion, uptake by targeted tissues and associated metabolic response. In this report, we describe the formation of microvascularised cardiac spheroids embedded in microfluidic chips. Different protocols used for embedding spheroids within vascularised multi-compartment microfluidic chips were investigated first to identify the importance of the spheroid processing, and co-culture with pericytes on the integration of the spheroid within the microvascular networks formed. The architecture of the resulting models, the expression of cardiac and endothelial markers and the perfusion of the system was then investigated. This confirmed the excellent stability of the vascular networks formed, as well as the persistent expression of cardiomyocyte markers such as cTNT and the assembly of striated F-actin, myosin and α-actinin cytoskeletal networks typically associated with contractility and beating. The ability to retain beating over prolonged periods of time was quantified, over 25 days, demonstrating not only perfusability but also functional performance of the tissue model. Finally, as a proof-of-concept of therapeutic testing, the toxicity of one therapeutic associated with cardiac disfunction was evaluated, identifying differences between direct in vitro testing on suspended spheroids and vascularised models

The impact of pericytes on the stability of microvascular networks in response to nanoparticles.

Recapitulating the normal physiology of the microvasculature is pivotal in the development of more complex in-vitro models and organ-on-chip designs. Pericytes are an important component of the vasculature, promoting vessel stability, inhibiting vascular permeability and maintaining the vascular hierarchical architecture. The use of such co-culture for the testing of therapeutics and nanoparticle safety is increasingly considered for the validation of therapeutic strategies. This report presents the use of a microfluidic model for such applications. Interactions between endothelial cells and pericytes are first explored. We identify basal conditions required to form stable and reproducible endothelial networks. We then investigate interactions between endothelial cells and pericytes via direct co-culture. In our system, pericytes prevented vessel hyperplasia and maintained vessel length in prolonged culture (> 10 days). In addition, these vessels displayed barrier function and expression of junction markers associated with vessel maturation, including VE-cadherin, β-catenin and ZO-1. Furthermore, pericytes maintained vessel integrity following stress (nutrient starvation) and prevented vessel regression, in contrast to the striking dissociation of networks in endothelial monocultures. This response was also observed when endothelial/pericyte co-cultures were exposed to high concentrations of moderately toxic cationic nanoparticles used for gene delivery. This study highlights the importance of pericytes in protecting vascular networks from stress and external agents and their importance to the design of advanced in-vitro models, including for the testing of nanotoxicity, to better recapitulate physiological response and avoid false positives