Why bioprinting in regenerative medicine should adopt a rational technology readiness assessment

Bioprinting is an annex of additive manufacturing, as defined by the American Society for Testing and Materials (ASTM) and International Organization for Standardization (ISO) standards, characterized by the automated deposition of living cells and biomaterials. The tissue engineering and regenerative medicine (TE&amp;RM) community has eagerly adopted bioprinting, while review articles regularly herald its imminent translation to the clinic as functional tissues and organs. Here we argue that such proclamations are premature and counterproductive; they place emphasis on technological progress while typically ignoring the critical stage-gates that must be passed through to bring a technology to market. We suggest the technology readiness level (TRL) scale as a valuable metric for gauging the relative maturity of a bioprinting technology in relation to how it has passed a series of key milestones. We suggest guidelines for a bioprinting-oriented scale and use this to discuss the state-of-the-art of bioprinting in regenerative medicine (BRM) today. Finally, we make corresponding recommendations for improvements to BRM research that would support its progression to clinical translation.</p

Vapour phase polymerization of PEDOT from micron and nanometer scale oxidant patterned by dip-pen nanolithography

Some of the most exciting recent advances in conducting polymer synthesis have centered around the method of vapor phase polymerization (VPP) of thin films. However, it is not known whether the VPP process can proceed using significantly reduced volumes of oxidant and therefore be implemented as part of nanolithography approach. Here, we present a strategy for submicrometer scale patterning of the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) via in situ VPP. Attolitre (10-18 L) volumes of oxidant "ink" are controllably deposited using dip-pen nanolithography (DPN). DPN patterning of the oxidant ink is facilitated by the incorporation of an amphiphilic block copolymer thickener, an additive that also assists with stabilization of the oxidant. When exposed to EDOT monomer in a VPP chamber, each deposited feature localizes the synthesis of conducting PEDOT structures of several micrometers down to 250 nm in width. PEDOT patterns are characterized by atomic force microscopy (AFM), conductive AFM, two probe electrical measurement, and micro-Raman spectroscopy, evidencing in situ vapor phase synthesis of conducting polymer at a scale (picogram) which is much smaller than that previously reported. Although the process of VPP on this scale was achieved, we highlight some of the challenges that need to be overcome to make this approach feasible in an applied setting

Ink-on-probe hydrodynamics in atomic force microscope deposition of liquid inks

The controlled deposition of attolitre volumes of liquids may engender novel applications such as soft, nano-tailored cell-material interfaces, multi-plexed nano-arrays for high throughput screening of biomolecular interactions, and localized delivery of reagents to reactions confined at the nano-scale. Although the deposition of small organic molecules from an AFM tip, known as dip-pen nanolithography (DPN), is being continually refined, AFM deposition of liquid inks is not well understood, and is often fraught with inconsistent deposition rates. In this work, the variation in feature-size over long term printing experiments for four model inks of varying viscosity is examined. A hierarchy of recurring phenomena is uncovered and there are attributed to ink movement and reorganisation along the cantilever itself. Simple analytical approaches to model these effects, as well as a method to gauge the degree of ink loading using the cantilever resonance frequency, are described. In light of the conclusions, the various parameters which need to be controlled in order to achieve uniform printing are dicussed. This work has implications for the nanopatterning of viscous liquids and hydrogels, encompassing ink development, the design of probes and printing protocols

Governed by technology? Urban management of broadband and 3G systems in Sweden

Due to the current interest in organic printable electronics, Dip-Pen Nanolithography (DPN) is increasingly being explored as a method to pattern electroactive materials such as conducting polymers (CPs) on the micro- and nanoscale. In general, printing process depends strongly on the ink-substrate properties and fundamental forces required to drive and stabilize the ink transfer to the substrate. Controlling these parameters is especially difficult when operating under the nanometer confinements of the DPN probe. For the printing of CPs, one step towards addressing these challenges is rational ink design, as the use of existing commercial-based inks may not be suitable for the DPN ink transfer process on the nanoscale. In this study, we synthesized and developed a poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS)-based ink, to which we could add known constituents for optimizing the printing, adhesive and conductive properties of an aqueous dispersed ink. For silicon and gold surfaces, we demonstrate that the DPN pattering of the ink could achieve PEDOT:PSS (dot) arrays with diameters ranging from the submicron down to ∼160 nm. These dimensions represent a dramatic improvement in resolution compared to previous attempts in patterning of CP via physioadsorption process using DPN. This work highlights that rational design of CP inks is critical, especially for DPN where the ink transfer process is governed by fluid physical properties and surface forces in the nanodomain. Knowledge of the constituents and overall ink composition will also lead to a greater understanding of these fundamentals that facilitate the pattering of CP inks on the nanoscale using DPN

A Versatile Method to Create Perfusable, Capillary‐Scale Channels in Cell‐Laden Hydrogels Using Melt Electrowriting

Abstract A major obstacle toward creating human‐scale artificial tissue models is supplying encapsulated cells with oxygen and other nutrients throughout the construct. In particular, creating channels in hydrogels that match the resolution and density of the smallest blood capillaries (≤10 µm) remains highly challenging. Here, a novel method is demonstrated where polycaprolactone fibers printed using melt‐electrowriting are encapsulated in cell‐laden hydrogels and then physically removed to produce hollow, perfusable channels. This technique produces a range of channel diameters (10–41 µm) with circular cross‐sections and in hydrogels representing various crosslinking mechanisms. The channels can be formed as interconnected grids, hierarchically branched patterns, or stacked in layers with ≈200 µm channel spacing, thus matching average capillary density in the human body. Alternatively, selective removal of fibers from a melt electrowriting grid can generate perfusable channels within a reinforcing fiber network. This method can be performed in the presence of cells, with human fibroblasts exhibiting encapsulated in gelatin methacryloyl showing no detectable cytotoxic effects. This technique is a promising approach for creating perfusable channels with very small diameters within cell‐laden hydrogel matrices, with potential applications including in vitro tissue models and hydrogel microfluidics

FLASH: Fluorescently LAbelled Sensitive Hydrogel to monitor bioscaffolds degradation during neocartilage generation

© 2020 Elsevier Ltd Regenerative therapies based on photocrosslinkable hydrogels and stem cells are of growing interest in the field of cartilage repair. Cell-mediated degradation is critical for the successful clinical translation of implanted hydrogels. However, characterising cell-mediated degradation, while simultaneously monitoring the deposition of a distinct new matrix, remains a major challenge. In this study we generated a Fluorescently LAbelled Sensitive Hydrogel (FLASH) to correlate the degradation of a hydrogel bioscaffold with neocartilage formation. Gelatine Methacryloyl (GelMA) was covalently bound to the FITC fluorophore to generate FLASH and bioscaffolds were produced by casting different concentrations of FLASH GelMA, with and without human adipose-derived stem cells (hADSCs) undergoing chondrogenesis. The loss of fluorescence from FLASH bioscaffolds was correlated with changes in mechanical properties, expression of chondrogenic markers and accumulation of a cartilaginous extracellular matrix. The ability of the system to be used as a sensor to monitor bioscaffold degradability during chondrogenesis was evaluated in vitro, in a human ex vivo model of cartilage repair and in a full chondral defect in vivo rabbit model. This study represents a step towards the generation of a high throughput monitoring system to evaluate de novo cartilage formation in tissue engineering therapies