3D Printed Multiphasic Scaffolds for Osteochondral Repair: Challenges and Opportunities.

Osteochondral (OC) defects are debilitating joint injuries characterized by the loss of full thickness articular cartilage along with the underlying calcified cartilage through to the subchondral bone. While current surgical treatments can provide some relief from pain, none can fully repair all the components of the OC unit and restore its native function. Engineering OC tissue is challenging due to the presence of the three distinct tissue regions. Recent advances in additive manufacturing provide unprecedented control over the internal microstructure of bioscaffolds, the patterning of growth factors and the encapsulation of potentially regenerative cells. These developments are ushering in a new paradigm of 'multiphasic' scaffold designs in which the optimal micro-environment for each tissue region is individually crafted. Although the adoption of these techniques provides new opportunities in OC research, it also introduces challenges, such as creating tissue interfaces, integrating multiple fabrication techniques and co-culturing different cells within the same construct. This review captures the considerations and capabilities in developing 3D printed OC scaffolds, including materials, fabrication techniques, mechanical function, biological components and design.

Controlled release from PCL-alginate microspheres via secondary encapsulation using GelMA/HAMA hydrogel scaffolds.

Controlling the release of bioactive agents has important potential applications in tissue engineering. While microspheres have been investigated to manipulate release rates, the majority of these investigations have been based on delivery into aqueous media, whereas the cellular environment in tissue engineering is more typically a hydrogel scaffold. If drug-loaded microspheres are introduced within scaffolds to deliver biologically active substances in situ, it is crucial to understand how the release rate is influenced by interactions between the microspheres and the scaffold. Here, we report the fabrication and characterization of a biodegradable scaffold that contains composite microspheres and is suitable for biological applications. Our approach evaluates the influence on the release profile of a model drug (FITC-dextran sulfate) from alginate and PCL-alginate microspheres within a hydrogel construct forming a secondary encapsulation. Increasing the degree of crosslinking in the secondary encapsulation matrix led to a slower cumulative release from 36% to 15%, from the alginate microspheres, whereas a decrease from 26% to 6% was observed for the PCL-alginate microspheres. These results suggest that the release of bioactive molecules can be fine tuned by independently engineering the properties of the scaffold and microspheres.

Tailoring the mechanical properties of gelatin methacryloyl hydrogels through manipulation of the photocrosslinking conditions.

Photo-crosslinkable hydrogels, in particular gelatin methacryloyl (GelMa), are gaining increasing importance in biofabrication and tissue engineering. While GelMa is often described as mechanically 'tunable', clear relationships linking the photocrosslinking conditions to reaction rates, and the resulting mechanical properties, have not been described. Meanwhile the conditions employed in the literature are disparate, and difficult to compare. In this work, in situ rheological measurements were used to quantify the relative rate of reaction of GelMa hydrogels with respect to light intensity, exposure time and photo-initiator concentration. In addition the UV degradation of the photo-initiator Irgacure 2959 was measured by UV-vis spectroscopy, and used to estimate the rate of free radical production as a function of light exposure. Using these data an expression was derived which predicts the mechanical properties of GelMa hydrogels produced across a wide range of crosslinking conditions. The model was validated through fabrication of a GelMa gradient which matched predicted properties. Human mesenchymal stem cells encapsulated in crosslinked GelMa exhibited high (>90%) viability post encapsulation, however metabolic activity over one week was influenced by the intensity of light used during crosslinking. The expressions described may be used to aid rational choices of GelMa photocrosslinking conditions, especially in cell encapsulation experiments where minimising the cytotoxic elements in the reaction is a priority.

Enthusiastic portrayal of 3D bioprinting in the media: Ethical side effects.

There has been a surge in mass media reports extolling the potential for using three-dimensional printing of biomaterials (3D bioprinting) to treat a wide range of clinical conditions. Given that mass media is recognized as one of the most important sources of health and medical information for the general public, especially prospective patients, we report and discuss the ethical consequences of coverage of 3D bioprinting in the media. First, we illustrate how positive mass media narratives of a similar biofabricated technology, namely the Macchiarini scaffold tracheas, which was involved in lethal experimental human trials, influenced potential patient perceptions. Second, we report and analyze the positively biased and enthusiastic portrayal of 3D bioprinting in mass media. Third, we examine the lack of regulation and absence of discussion about risks associated with bioprinting technology. Fourth, we explore how media misunderstanding is dangerously misleading the narrative about the technology.

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.

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.

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.

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&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.

Miniature fluorescence sensor for quantitative detection of brain tumour.

Fluorescence-guided surgery has emerged as a vital tool for tumour resection procedures. As well as intraoperative tumour visualisation, 5-ALA-induced PpIX provides an avenue for quantitative tumour identification based on ratiometric fluorescence measurement. To this end, fluorescence imaging and fibre-based probes have enabled more precise demarcation between the cancerous and healthy tissues. These sensing approaches, which rely on collecting the fluorescence light from the tumour resection site and its "remote" spectral sensing, introduce challenges associated with optical losses. In this work, we demonstrate the viability of tumour detection at the resection site using a miniature fluorescence measurement system. Unlike the current bulky systems, which necessitate remote measurement, we have adopted a millimetre-sized spectral sensor chip for quantitative fluorescence measurements. A reliable measurement at the resection site requires a stable optical window between the tissue and the optoelectronic system. This is achieved using an antifouling diamond window, which provides stable optical transparency. The system achieved a sensitivity of 92.3% and specificity of 98.3% in detecting a surrogate tumour at a resolution of 1 × 1 mm2. As well as addressing losses associated with collecting and coupling fluorescence light in the current 'remote' sensing approaches, the small size of the system introduced in this work paves the way for its direct integration with the tumour resection tools with the aim of more accurate interoperative tumour identification.

Engineering interfacial tissues: The myotendinous junction.

The myotendinous junction (MTJ) is the interface connecting skeletal muscle and tendon tissues. This specialized region represents the bridge that facilitates the transmission of contractile forces from muscle to tendon, and ultimately the skeletal system for the creation of movement. MTJs are, therefore, subject to high stress concentrations, rendering them susceptible to severe, life-altering injuries. Despite the scarcity of knowledge obtained from MTJ formation during embryogenesis, several attempts have been made to engineer this complex interfacial tissue. These attempts, however, fail to achieve the level of maturity and mechanical complexity required for in vivo transplantation. This review summarizes the strategies taken to engineer the MTJ, with an emphasis on how transitioning from static to mechanically inducive dynamic cultures may assist in achieving myotendinous maturity.

Negative Printing for the Reinforcement of In Situ Tissue-Engineered Cartilage.

In the realm of in situ cartilage engineering, the targeted delivery of both cells and hydrogel materials to the site of a defect serves to directly stimulate chondral repair. Although the in situ application of stem cell-laden soft hydrogels to tissue defects holds great promise for cartilage regeneration, a significant challenge lies in overcoming the inherent limitation of these soft hydrogels, which must attain mechanical properties akin to the native tissue to withstand physiological loading. We therefore developed a system where a gelatin methacryloyl hydrogel laden with human adipose-derived mesenchymal stem cells is combined with a secondary structure to provide bulk mechanical reinforcement. In this study, we used the negative embodied sacrificial template 3D printing technique to generate eight different lattice-based reinforcement structures made of polycaprolactone, which ranged in porosity from 80% to 90% with stiffnesses from 28 ± 5 kPa to 2853 ± 236 kPa. The most promising of these designs, the hex prism edge, was combined with the cellular hydrogel and retained a stable stiffness over 41 days of chondrogenic differentiation. There was no significant difference between the hydrogel-only and hydrogel scaffold group in the sulfated glycosaminoglycan production (340.46 ± 13.32 µg and 338.92 ± 47.33 µg, respectively) or Type II Collagen gene expression. As such, the use of negative printing represents a promising solution for the integration of bulk reinforcement without losing the ability to produce new chondrogenic matrix.

NEST3D printed bone-mimicking scaffolds: assessment of the effect of geometrical design on stiffness and angiogenic potential.

Tissue-engineered implants for bone regeneration require consideration regarding their mineralization and vascularization capacity. Different geometries, such as biomimetic designs and lattices, can influence the mechanical properties and the vascularization capacity of bone-mimicking implants. Negative Embodied Sacrificial Template 3D (NEST3D) printing is a versatile technique across a wide range of materials that enables the production of bone-mimicking scaffolds. In this study, different scaffold motifs (logpile, Voronoi, and trabecular bone) were fabricated via NEST3D printing in polycaprolactone to determine the effect of geometrical design on stiffness (10.44 ± 6.71, 12.61 ± 5.71, and 25.93 ± 4.16 MPa, respectively) and vascularization. The same designs, in a polycaprolactone scaffold only, or when combined with gelatin methacryloyl, were then assessed for their ability to allow the infiltration of blood vessels in a chick chorioallantoic membrane (CAM) assay, a cost-effective and time-efficient in ovo assay to assess vascularization. Our findings showed that gelatin methacrylolyl alone did not allow new chorioallantoic membrane tissue or blood vessels to infiltrate within its structure. However, polycaprolactone on its own or when combined with gelatin methacrylolyl allowed tissue and vessel infiltration in all scaffold designs. The trabecular bone design showed the greatest mineralized matrix production over the three designs tested. This reinforces our hypothesis that both biomaterial choice and scaffold motifs are crucial components for a bone-mimicking scaffold.

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.

Towards Clinical Translation of In Situ Cartilage Engineering Strategies: Optimizing the Critical Facets of a Cell-Laden Hydrogel Therapy.

BACKGROUND: Articular cartilage repair using implantable photocrosslinkable hydrogels laden with chondrogenic cells, represents a promising in situ cartilage engineering approach for surgical treatment. The development of a surgical procedure requires a minimal viable product optimized for the clinical scenario. In our previous work we demonstrated how gelatin based photocrosslinkable hydrogels in combination with infrapatellar derived stem cells allow the production of neocartilage in vitro. In this study, we aim to optimize the critical facets of the in situ cartilage engineering therapy: the cell source, the cell isolation methodology, the cell expansion protocol, the cell number, and the delivery approach. METHODS: We evaluated the impact of the critical facets of the cell-laden hydrogel therapy in vitro to define an optimized protocol that was then used in a rabbit model of cartilage repair. We performed cells counting and immunophenotype analyses, chondrogenic potential evaluation via immunostaining and gene expression, extrusion test analysis of the photocrosslinkable hydrogel, and clinical assessment of cartilage repair using macroscopic and microscopic scores. RESULTS: We identified the adipose derived stem cells as the most chondrogenic cells source within the knee joint. We then devised a minimally manipulated stem cell isolation procedure that allows a chondrogenic population to be obtained in only 85 minutes. We found that cell expansion prior to chondrogenesis can be reduced to 5 days after the isolation procedure. We characterized that at least 5 million of cells/ml is needed in the photocrosslinkable hydrogel to successfully trigger the production of neocartilage. The maximum repairable defect was calculated based on the correlation between the number of cells retrievable with the rapid isolation followed by 5-day non-passaged expansion phase, and the minimum chondrogenic concentration in photocrosslinkable hydrogel. We next optimized the delivery parameters of the cell-laden hydrogel therapy. Finally, using the optimized procedure for in situ tissue engineering, we scored superior cartilage repair when compared to the gold standard microfracture approach. CONCLUSION: This study demonstrates the possibility to repair a critical size articular cartilage defect by means of a surgical streamlined procedure with optimized conditions.

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.

Gelatin acrylamide with improved UV crosslinking and mechanical properties for 3D biofabrication.

Photocrosslinkable gelatin has attracted increasing interest in the field of biofabrication, with the most studied and widely used photocrosslinkable gelatin being gelatin methacrylate (GelMa). However, the 3D fabrication of GelMa has presented several limitations and challenges, primarily due to its slow crosslinking speed. It is generally known that acryl-based functional groups have faster reaction kinetics than methacryl-base groups. However, gelatin acrylamide (GelAc) has not been widely investigated, largely due to its increased complexity of synthesis relative to GelMA. In this study, we developed a novel synthesis method for GelAc. By varying the reaction ratio of reagents, GelAc with a degree of substitution from 20% to 95% was produced. The UV crosslinking properties of GelAc was studied, demonstrating significantly faster crosslinking kinetics than GelMa, especially at lower concentrations and low photoinitiator concentrations. The swelling ratio and mechanical properties of the crosslinked GelAc hydrogel were also characterized, and biocompatibility experiments conducted via both surface seeding and hydrogel encapsulation of cells, with good cell viability observed. The application of GelAc for 3D biofabrication was demonstrated by 3D printing. GelAc can be a useful material for the fabrication of 3D conduits for tissue engineering applications.

Layer-by-Layer Analysis of In Vitro Skin Models.

In vitro human skin models are evolving into versatile platforms for the study of skin biology and disorders. These models have many potential applications in the fields of drug testing and safety assessment, as well as cosmetic and new treatment development. The development of in vitro skin models that accurately mimic native human skin can reduce reliance on animal models and also allow for more precise, clinically relevant testing. Recent advances in biofabrication techniques and biomaterials have led to the creation of increasingly complex, multilayered skin models that incorporate important functional components of skin, such as the skin barrier, mechanical properties, pigmentation, vasculature, hair follicles, glands, and subcutaneous layer. This improved ability to recapitulate the functional aspects of native skin enhances the ability to model the behavior and response of native human skin, as the complex interplay of cell-to-cell and cell-to-material interactions are incorporated. In this review, we summarize the recent developments in in vitro skin models, with a focus on their applications, limitations, and future directions.

Liquid deposition patterning of conducting polymer ink onto hard and soft flexible substrates via dip-pen nanolithography.

Ink formulations and protocols that enable the deposition and patterning of a conducting polymer (PEDOT:PSS) in the nanodomain have been developed. Significantly, we demonstrated the ability to pattern onto soft substrates such as silicone gum and polyethylene terephthalate (PET), which are materials of interest for low cost, flexible electronics. The deposition process and dimensions of the polymer patterns are found to be critically dependent on a number of parameters, including the pen design, ink properties, time after inking the pen, dwell time of the pen on the surface, and the nature of material substrate. By assessing these different parameters, an improved understanding of the ability to control the dimensions of individual PEDOT:PSS structures down to 600 nm in width and 10-80 nm in height within patterned arrays was obtained. This applicability of DPN for simple and nonreactive liquid deposition patterning of conducting polymers can lead to the fabrication of organic nanoelectronics or biosensors and complement the efforts of existing printing techniques such as inkjet and extrusion printing by scaling down conductive components to submicrometer and nanoscale dimensions.

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.

Within or Without You? A Perspective Comparing In Situ and Ex Situ Tissue Engineering Strategies for Articular Cartilage Repair.

Human articular cartilage has a poor ability to self-repair, meaning small injuries often lead to osteoarthritis, a painful and debilitating condition which is a major contributor to the global burden of disease. Existing clinical strategies generally do not regenerate hyaline type cartilage, motivating research toward tissue engineering solutions. Prospective cartilage tissue engineering therapies can be placed into two broad categories: i) Ex situ strategies, where cartilage tissue constructs are engineered in the lab prior to implantation and ii) in situ strategies, where cells and/or a bioscaffold are delivered to the defect site to stimulate chondral repair directly. While commonalities exist between these two approaches, the core point of distinction-whether chondrogenesis primarily occurs "within" or "without" (outside) the body-can dictate many aspects of the treatment. This difference influences decisions around cell selection, the biomaterials formulation and the surgical implantation procedure, the processes of tissue integration and maturation, as well as, the prospects for regulatory clearance and clinical translation. Here, ex situ and in situ cartilage engineering strategies are compared: Highlighting their respective challenges, opportunities, and prospects on their translational pathways toward long term human cartilage repair.