Biofabrication of human articular cartilage: a path towards the development of a clinical treatment

Cartilage injuries cause pain and loss of function, and if severe may result in osteoarthritis (OA). 3D bioprinting is now a tangible option for the delivery of bioscaffolds capable of regenerating the deficient cartilage tissue. Our team has developed a handheld device, the Biopen, to allow in situ additive manufacturing during surgery. Given its ability to extrude in a core/shell manner, the Biopen can preserve cell viability during the biofabrication process, and it is currently the only biofabrication tool tested as a surgical instrument in a sheep model using homologous stem cells. As a necessary step toward the development of a clinically relevant protocol, we aimed to demonstrate that our handheld extrusion device can successfully be used for the biofabrication of human cartilage. Therefore, this study is a required step for the development of a surgical treatment in human patients. In this work we specifically used human adipose derived mesenchymal stem cells (hADSCs), harvested from the infrapatellar fat pad of donor patients affected by OA, to also prove that they can be utilized as the source of cells for the future clinical application. With the Biopen, we generated bioscaffolds made of hADSCs laden in gelatin methacrylate, hyaluronic acid methacrylate and cultured in the presence of chondrogenic stimuli for eight weeks in vitro. A comprehensive characterisation including gene and protein expression analyses, immunohistology, confocal microscopy, second harmonic generation, light sheet imaging, atomic force mycroscopy and mechanical unconfined compression demonstrated that our strategy resulted in human hyaline-like cartilage formation. Our in situ biofabrication approach represents an innovation with important implications for customizing cartilage repair in patients with cartilage injuries and OA

Handheld Co-Axial Bioprinting: Application to in situ surgical cartilage repair

Three-dimensional (3D) bioprinting is driving major innovations in the area of cartilage tissue engineering. Extrusion-based 3D bioprinting necessitates a phase change from a liquid bioink to a semi-solid crosslinked network achieved by a photo-initiated free radical polymerization reaction that is known to be cytotoxic. Therefore, the choice of the photocuring conditions has to be carefully addressed to generate a structure stiff enough to withstand the forces phisiologically applied on articular cartilage, while ensuring adequate cell survival for functional chondral repair. We recently developed a handheld 3D printer called Biopen . To progress towards translating this freeform biofabrication tool into clinical practice, we aimed to define the ideal bioprinting conditions that would deliver a scaffold with high cell viability and structural stiffness relevant for chondral repair. To fulfill those criteria, free radical cytotoxicity was confined by a co-axial Core/Shell separation. This system allowed the generation of Core/Shell GelMa/HAMa bioscaffolds with stiffness of 200KPa, achieved after only 10seconds of exposure to 700mW/cm2 of 365nm UV-A, containing \u3e90% viable stem cells that retained proliferative capacity. Overall, the Core/Shell handheld 3D bioprinting strategy enabled rapid generation of high modulus bioscaffolds with high cell viability, with potential for in situ surgical cartilage engineering

Novel strategies for depositing nanoelectrode materials using dip-pen nanolithography of liquid inks

Medical bionic devices restore human function by interfacing electrical technology with the body. The emerging field of nanobionics is borne from advances in our ability to control the structure of materials on finer and finer length-scales, coupled with an increased appreciation of the sensitivity of living cells to nanoscale topographical, chemical and mechanical cues. As we envisage and prototype nanostructured bionic devices there is a crucial need to understand how cells feel and respond to nanoscale materials, particularly as material properties (surface energy, conductivity etc.) can be very different at the nanoscale than at bulk. However, the patterning of bionic materials of interest is often not achievable using conventional fabrication techniques, especially on soft, biocompatible substrates. Nonconventional nanofabrication strategies are required. Dip-pen nanolithography (DPN) is a nanofabrication technique which uses the nanoscale tip of an atomic force microscope to direct-write functional materials. This thesis contributes to the development of the DPN technique in two main aspects. The first aspect is the development of two novel methods of patterning electro-materials at submicron- to nano resolution. The second is a contribution to the understanding of ink transport in liquid ink DPN. A novel oxidant ink was developed for in situ synthesis of the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) via vapour phase polymerisation. DPN patterning of the oxidant ink was facilitated by the incorporation of an amphiphilic block copolymer thickener, an additive that also assisted with stabilization of the oxidant. When exposed to EDOT monomer in a VPP chamber, each deposited feature localized the synthesis of conducting PEDOT structures of several micro-meters down to 250 nm in width, at a scale (picogram) which is much smaller than any previously reported. A strategy was developed to DPN print a platinum precursor (H2PtCl6) based liquid ink onto insulating substrates with nanoscale resolution. The ink formulation was printable on Si, glass, ITO, Ge, PDMS, Parylene C and even a human hair. A mild plasma treatment effected reduction of the precursor patterns in situ without subjugating the substrate to destructively high temperatures. Feature size was controlled via dwell time and degree of ink loading, and platinum features with 50 nm dimensions could be routinely achieved on silanized Si. We confirmed the electrical conductivity of printed platinum by two point probe measurements and we characterized electrochemical activity using Scanning Electrochemical Microscopy (SECM). A modified method enabled deposition of micron scale Pt snowflakes. By tuning the substrate hydrophobicity using functionalization with a long chain alkane group the spreading of the precursor ink was tempered, and growth of a fractal-like crystal proceeded via a diffusion limited aggregation mechanism. Reduction of the precursor crystal by plasma treatment resulted in a 2D dendritic structure composed of Pt nanoparticles. This combined top-down/bottom-up approach enabled the arbitrary placement of \u3c 20 nm Pt fractal-like structures on Si or glass. Model ink-substrate systems, which exhibiting a range of viscosities and wettabilities, were used to explore various methods of controlling feature size in liquid ink DPN. The ink-transfer mechanism was investigated using AFM force measurements acquired during ink deposition. This data was used to elucidate the shape of the meniscus during deposition and illustrate a method to monitor the volume of deposition in-situ. However, the deposition rate was found to change dramatically over the course of an experiment due to a dependence of deposition rate on the changing volume of ink on the pen. The effect of depleting ink volume on deposition rate over a long term experiment was investigated. A hierarchy of phenomena were uncovered which were related to ink movement and reorganisation along the cantilever. These ‘ink-on-tip hydrodynamics’ were suggested to arise from (I) changes in ink volume on cantilever, (II) ‘rest-time’ between grids and (III) travel time between individual dots. In light of our conclusions, we posed critical questions of reservoir-on-tip liquid ink DPN as a nanofabrication technique and discuss the various parameters which need to be controlled in order to achieve uniformity of feature size. The novel electro-material printing strategies developed in this thesis may have applications in the fabrication of nanoelectronic and nanobionic platforms, particularly on flexible, polymeric substrates. The insights gained into the dynamics of liquid ink transport may have implications in the rational design of DPN inks and probes

Layer-by-layer: The case for 3D bioprinting neurons to create patient-specific epilepsy models

The ability to create three-dimensional (3D) models of brain tissue from patient-derived cells, would open new possibilities in studying the neuropathology of disorders such as epilepsy and schizophrenia. While organoid culture has provided impressive examples of patient-specific models, the generation of organised 3D structures remains a challenge. 3D bioprinting is a rapidly developing technology where living cells, encapsulated in suitable bioink matrices, are printed to form 3D structures. 3D bioprinting may provide the capability to organise neuronal populations in 3D, through layer-by-layer deposition, and thereby recapitulate the complexity of neural tissue. However, printing neuron cells raises particular challenges since the biomaterial environment must be of appropriate softness to allow for the neurite extension, properties which are anathema to building self-supporting 3D structures. Here, we review the topic of 3D bioprinting of neurons, including critical discussions of hardware and bio-ink formulation requirements

Vapor phase polymerization of EDOT from submicrometer 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

Liquid ink deposition from an atomic force microscope tip: deposition monitoring and control of feature size

The controlled deposition of attoliter volumes of liquid inks may engender novel applications such as targeted drug delivery to single cells and localized delivery of chemical reagents at nanoscale dimensions. Although the deposition of small organic molecules from an atomic force microscope tip, known as dip-pen nanolithography (DPN), has been extensively studied, the deposition of liquid inks is little understood. In this work, we have used a set of model ink-substrate systems to develop an understanding of the deposition of viscous liquids using an unmodified AFM tip. First, the growth of dot size with increasing dwell time is characterized. The dynamics of deposition are found to vary for different ink-substrate systems, and the change in deposition rate over the course of an experiment limits our ability to quantify the ink-transfer dynamics in terms of liquid properties and substrate wettability. We find that the most critical parameter affecting the deposition rate is the volume of ink on the cantilever, an effect resulting in a 10-fold decrease in deposition rate (aL/s) over 2 h of printing time. We suggest that a driving force for deposition arises from the gradient in Laplace pressure set up when the tip touches the substrate. Second, the forces acting upon the AFM cantilever during ink deposition were measured in order to gain insight into the underlying ink-transfer mechanism. The force curve data and simple geometrical arguments were used to elucidate the shape of the ink meniscus at the instant of deposition, a methodology that may be used as an accurate and real-time means of monitoring the volume of deposited dots. Taken together, our results illustrate that liquid deposition involves a very different transfer mechanism than traditionally ascribed to DPN molecular transport

Print Me an Organ? Ethical and Regulatory Issues Emerging from 3D Bioprinting in Medicine

Recent developments of three-dimensional printing of biomaterials (3D bioprinting) in medicine have been portrayed as demonstrating the potential to transform some medical treatments, including providing new responses to organ damage or organ failure. However, beyond the hype and before 3D bioprinted organs are ready to be transplanted into humans, several important ethical concerns and regulatory questions need to be addressed. This article starts by raising general ethical concerns associated with the use of bioprinting in medicine, then it focuses on more particular ethical issues related to experimental testing on humans, and the lack of current international regulatory directives to guide these experiments. Accordingly, this article (1) considers whether there is a limit as to what should be bioprinted in medicine; (2) examines key risks of significant harm associated with testing 3D bioprinting for humans; (3) investigates the clinical trial paradigm used to test 3D bioprinting; (4) analyses ethical questions of irreversibility, loss of treatment opportunity and replicability; (5) explores the current lack of a specific framework for the regulation and testing of 3D bioprinting treatments

The regulatory challenge of 3D bioprinting

New developments in additive manufacturing and regenerative medicine have the potential to radically disrupt the traditional pipelines of therapy development and medical device manufacture. These technologies present a challenge for regulators because traditional regulatory frameworks are designed for mass manufactured therapies, rather than bespoke solutions. 3D bioprinting technologies present another dimension of complexity through the inclusion of living cells in the fabrication process. Herein we overview the challenge of regulating 3D bioprinting in comparison to existing cell therapy products as well as custom-made 3D printed medical devices. We consider a range of specific challenges pertaining to 3D bioprinting in regenerative medicine, including classification, risk, standardization and quality control, as well as technical issues related to the manufacturing process and the incorporated materials and cells

Optimizing the composition of gelatin methacryloyl and hyaluronic acid methacryloyl hydrogels to maximize mechanical and transport properties using response surface methodology

Hydrogel materials are promising candidates in cartilage tissue engineering as they provide a 3D porous environment for cell proliferation and the development of new cartilage tissue. Both the mechanical and transport properties of hydrogel scaffolds influence the ability of encapsulated cells to produce neocartilage. In photocrosslinkable hydrogels, both of these material properties can be tuned by changing the crosslinking density. However, the interdependent nature of the structural, physical and biological properties of photocrosslinkable hydrogels means that optimizing composition is typically a complicated process, involving sequential and/or iterative steps of physiochemical and biological characterization. The combinational nature of the variables indicates that an exhaustive analysis of all reasonable concentration ranges would be impractical. Herein, response surface methodology (RSM) was used to efficiently optimize the composition of a hybrid of gelatin-methacryloyl (GelMA) and hyaluronic acid methacryloyl (HAMA) with respect to both mechanical and transport properties. RSM was employed to investigate the effect of GelMA, HAMA, and photoinitiator concentration on the shear modulus and diffusion coefficient of the hydrogel membrane. Two mathematical models were fitted to the experimental data and used to predict the optimum hydrogel composition. Finally, the optimal composition was tested and compared with the predicted values