Digitally Driven Aerosol Jet Printing to Enable Customisable Neuronal Guidance.

Digitally driven manufacturing technologies such as aerosol jet printing (AJP) can make a significant contribution to enabling new capabilities in the field of tissue engineering disease modeling and drug screening. AJP is an emerging non-contact and mask-less printing process which has distinct advantages over other patterning technologies as it offers versatile, high-resolution, direct-write deposition of a variety of materials on planar and non-planar surfaces. This research demonstrates the ability of AJP to print digitally controlled patterns that influence neuronal guidance. These consist of patterned poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) tracks on both glass and poly(potassium 3-sulfopropyl methacrylate) (PKSPMA) coated glass surfaces, promoting selective adhesion of SH-SY5Y neuroblastoma cells. The cell attractive patterns had a maximum height ≥0.2 µm, width and half height ≥15 µm, Ra = 3.5 nm, and RMS = 4.1. The developed biocompatible PEDOT:PSS ink was shown to promote adhesion, growth and differentiation of SH-SY5Y neuronal cells. SH-SY5Y cells cultured directly onto these features exhibited increased nuclei and neuronal alignment on both substrates. In addition, the cell adhesion to the substrate was selective when cultured onto the PKSPMA surfaces resulting in a highly organized neural pattern. This demonstrated the ability to rapidly and flexibly realize intricate and accurate cell patterns by a computer controlled process.

Bioengineered model of the human motor unit with physiologically functional neuromuscular junctions.

Investigations of the human neuromuscular junction (NMJ) have predominately utilised experimental animals, model organisms, or monolayer cell cultures that fail to represent the physiological complexity of the synapse. Consequently, there remains a paucity of data regarding the development of the human NMJ and a lack of systems that enable investigation of the motor unit. This work addresses this need, providing the methodologies to bioengineer 3D models of the human motor unit. Spheroid culture of iPSC derived motor neuron progenitors augmented the transcription of OLIG2, ISLET1 and SMI32 motor neuron mRNAs ~ 400, ~ 150 and ~ 200-fold respectively compared to monolayer equivalents. Axon projections of adhered spheroids exceeded 1000 µm in monolayer, with transcription of SMI32 and VACHT mRNAs further enhanced by addition to 3D extracellular matrices in a type I collagen concentration dependent manner. Bioengineered skeletal muscles produced functional tetanic and twitch profiles, demonstrated increased acetylcholine receptor (AChR) clustering and transcription of MUSK and LRP4 mRNAs, indicating enhanced organisation of the post-synaptic membrane. The number of motor neuron spheroids, or motor pool, required to functionally innervate 3D muscle tissues was then determined, generating functional human NMJs that evidence pre- and post-synaptic membrane and motor nerve axon co-localisation. Spontaneous firing was significantly elevated in 3D motor units, confirmed to be driven by the motor nerve via antagonistic inhibition of the AChR. Functional analysis outlined decreased time to peak twitch and half relaxation times, indicating enhanced physiology of excitation contraction coupling in innervated motor units. Our findings provide the methods to maximise the maturity of both iPSC motor neurons and primary human skeletal muscle, utilising cell type specific extracellular matrices and developmental timelines to bioengineer the human motor unit for the study of neuromuscular junction physiology.

Physiological and pathophysiological concentrations of fatty acids induce lipid droplet accumulation and impair functional performance of tissue engineered skeletal muscle.

Fatty acids (FA) exert physiological and pathophysiological effects leading to changes in skeletal muscle metabolism and function, however, in vitro models to investigate these changes are limited. These experiments sought to establish the effects of physiological and pathophysiological concentrations of exogenous FA upon the function of tissue engineered skeletal muscle (TESkM). Cultured initially for 14 days, C2C12 TESkM was exposed to FA-free bovine serum albumin alone or conjugated to a FA mixture (oleic, palmitic, linoleic, and α-linoleic acids [OPLA] [ratio 45:30:24:1%]) at different concentrations (200 or 800 µM) for an additional 4 days. Subsequently, TESkM morphology, functional capacity, gene expression and insulin signaling were analyzed. There was a dose response increase in the number and size of lipid droplets within the TESkM (p < .05). Exposure to exogenous FA increased the messenger RNA expression of genes involved in lipid storage (perilipin 2 [p < .05]) and metabolism (pyruvate dehydrogenase lipoamide kinase isozyme 4 [p < .01]) in a dose dependent manner. TESkM force production was reduced (tetanic and single twitch) (p < .05) and increases in transcription of type I slow twitch fiber isoform, myosin heavy chain 7, were observed when cultured with 200 µM OPLA compared to control (p < .01). Four days of OPLA exposure results in lipid accumulation in TESkM which in turn results in changes in muscle function and metabolism; thus, providing insight ito the functional and mechanistic changes of TESkM in response to exogenous FA.

Functional regeneration of tissue engineered skeletal muscle in vitro is dependent on the inclusion of basement membrane proteins.

Skeletal muscle has a high regenerative capacity, injuries trigger a regenerative program which restores tissue function to a level indistinguishable to the pre-injury state. However, in some cases where significant trauma occurs, such as injuries seen in military populations, the regenerative process is overwhelmed and cannot restore full function. Limited clinical interventions exist which can be used to promote regeneration and prevent the formation of non-regenerative defects following severe skeletal muscle trauma. Robust and reproducible techniques for modelling complex tissue responses are essential to promote the discovery of effective clinical interventions. Tissue engineering has been highlighted as an alternative method, allowing the generation of three-dimensional in vivo like tissues without laboratory animals. Reducing the requirement for animal models promotes rapid screening of potential clinical interventions, as these models are more easily manipulated, genetically and pharmacologically, and reduce the associated cost and complexity, whilst increasing access to models for laboratories without animal facilities. In this study, an in vitro chemical injury using barium chloride is validated using the C2C12 myoblast cell line, and is shown to selectively remove multinucleated myotubes, whilst retaining a regenerative mononuclear cell population. Monolayer cultures showed limited regenerative capacity, with basement membrane supplementation or extended regenerative time incapable of improving the regenerative response. Conversely tissue engineered skeletal muscles, supplemented with basement membrane proteins, showed full functional regeneration, and a broader in vivo like inflammatory response. This work outlines a freely available and open access methodology to produce a cell line-based tissue engineered model of skeletal muscle regeneration.

Scalable 3D Printed Molds for Human Tissue Engineered Skeletal Muscle.

Tissue engineered skeletal muscle allows investigation of the cellular and molecular mechanisms that regulate skeletal muscle pathology. The fabricated model must resemble characteristics of in vivo tissue and incorporate cost-effective and high content primary human tissue. Current models are limited by low throughput due to the complexities associated with recruiting tissue donors, donor specific variations, as well as cellular senescence associated with passaging. This research presents a method using fused deposition modeling (FDM) and laser sintering (LS) 3D printing to generate reproducible and scalable tissue engineered primary human muscle, possessing aligned mature myotubes reminiscent of in vivo tissue. Many existing models are bespoke causing variability when translated between laboratories. To this end, a scalable model has been developed (25-500 µL construct volumes) allowing fabrication of mature primary human skeletal muscle. This research provides a strategy to overcome limited biopsy cell numbers, enabling high throughput screening of functional human tissue.

Polydimethylsiloxane and poly(ether) ether ketone functionally graded composites for biomedical applications.

Functionally graded materials (FGMs), with varying spatial, chemical and mechanical gradients (continuous or stepwise), have the potential to mimic heterogenous properties found across biological tissues. They can prevent stress concentrations and retain healthy cellular functions. Here, we show for the first time the fabrication of polydimethylsiloxane and poly(ether) ether ketone (PDMS-PEEK) composites. These were successfully manufactured as a bulk material and functionally graded (stepwise) without the use of hazardous solvents or the need of additives. Chemical, irreversible adhesion between layers (for the FGMs) was achieved without the formation of hard, boundary interfaces. The mechanical properties of PDMS-PEEK FGMs are proven to be further tailorable across the entirety of the build volume, mimicking the transition from soft to harder tissues. The introduction of 20 wt% PEEK particles into the PDMS matrix resulted in significant rises in the elastic modulus under tensile and compressive loading. Biological and thermal screenings suggested that these composites cause no adverse effects to human fibroblast cell lines and can retain physical state and mass at body temperature, which could make the composites suitable for a range of biomedical applications such as maxillofacial prosthetics, artificial blood vessels and articular cartilage replacement.

Differentiation of Bioengineered Skeletal Muscle within a 3D Printed Perfusion Bioreactor Reduces Atrophic and Inflammatory Gene Expression.

Bioengineered skeletal muscle tissues benefit from dynamic culture environments which facilitate the appropriate provision of nutrients and removal of cellular waste products. Biologically compatible perfusion systems hold the potential to enhance the physiological biomimicry of in vitro tissues via dynamic culture, in addition to providing technological advances in analytical testing and live cellular imaging for analysis of cellular development. To meet such diverse requirements, perfusion systems require the capacity and adaptability to incorporate multiple cell laden constructs of both monolayer and bioengineered tissues. This work reports perfusion systems produced using additive manufacturing technology for the in situ phenotypic development of myogenic precursor cells in monolayer and bioengineered tissue. Biocompatibility of systems 3D printed using stereolithography (SL), laser sintering (LS), and PolyJet outlined preferential morphological development within both SL and LS devices. When exposed to intermittent perfusion in the monolayer, delayed yet physiologically representative cellular proliferation, MyoD and myogenin transcription of C2C12 cells was evident. Long-term (8 days) intermittent perfusion of monolayer cultures outlined viable morphological and genetic in situ differentiation for the live cellular imaging of myogenic development. Continuous perfusion cultures (13 days) of bioengineered skeletal muscle tissues outlined in situ myogenic differentiation, forming mature multinucleated myotubes. Here, reductions in IL-1ß and TNF-α inflammatory cytokines, myostatin, and MuRF-1 atrophic mRNA expression were observed. Comparable myosin heavy chain (MyHC) isoform transcription profiles were evident between conditions; however, total mRNA expression was reduced in perfusion conditions. Decreased transcription of MuRF1 and subsequent reduced ubiquitination of the MyHC protein allude to a decreased requirement for transcription of MyHC isoform transcripts. Together, these data appear to indicate that 3D printed perfusion systems elicit enhanced stability of the culture environment, resulting in a reduced basal requirement for MyHC gene expression within bioengineered skeletal muscle tissue.

Feasibility and Biocompatibility of 3D-Printed Photopolymerized and Laser Sintered Polymers for Neuronal, Myogenic, and Hepatic Cell Types.

The integration of additive manufacturing (AM) technology within biological systems holds significant potential, specifically when refining the methods utilized for the creation of in vitro models. Therefore, examination of cellular interaction with the physical/physicochemical properties of 3D-printed polymers is critically important. In this work, skeletal muscle (C2 C12 ), neuronal (SH-SY5Y) and hepatic (HepG2) cell lines are utilized to ascertain critical evidence of cellular behavior in response to 3D-printed candidate polymers: Clear-FL (stereolithography, SL), PA-12 (laser sintering, LS), and VeroClear (PolyJet). This research outlines initial critical evidence for a framework of polymer/AM process selection when 3D printing biologically receptive scaffolds, derived from industry standard, commercially available AM instrumentation. C2 C12 , SH-SY5Y, and HepG2 cells favor LS polymer PA-12 for applications in which cellular adherence is necessitated. However, cell type specific responses are evident when cultured in the chemical leachate of photopolymers (Clear-FL and VeroClear). With the increasing prevalence of 3D-printed biointerfaces, the development of rigorous cell type specific biocompatibility data is imperative. Supplementing the currently limited database of functional 3D-printed biomaterials affords the opportunity for experiment-specific AM process and polymer selection, dependent on biological application and intricacy of design features required.

Biocompatible 3D printed polymers via fused deposition modelling direct C2C12 cellular phenotype in vitro.

The capability to 3D print bespoke biologically receptive parts within short time periods has driven the growing prevalence of additive manufacture (AM) technology within biological settings, however limited research concerning cellular interaction with 3D printed polymers has been undertaken. In this work, we used skeletal muscle C2C12 cell line in order to ascertain critical evidence of cellular behaviour in response to multiple bio-receptive candidate polymers; polylactic acid (PLA), acrylonitrile butadiene styrene (ABS), polyethylene terephthalate (PET) and polycarbonate (PC) 3D printed via fused deposition modelling (FDM). The extrusion based nature of FDM elicited polymer specific topographies, within which C2C12 cells exhibited reduced metabolic activity when compared to optimised surfaces of tissue culture plastic, however assay viability readings remained high across polymers outlining viable phenotypes. C2C12 cells exhibited consistently high levels of morphological alignment across polymers, however differential myotube widths and levels of transcriptional myogenin expression appeared to demonstrate response specific thresholds at which varying polymer selection potentiates cellular differentiation, elicits pre-mature early myotube formation and directs subsequent morphological phenotype. Here we observed biocompatible AM polymers manufactured via FDM, which also appear to hold the potential to simultaneously manipulate the desired biological phenotype and enhance the biomimicry of skeletal muscle cells in vitro via AM polymer choice and careful selection of machine processing parameters. When considered in combination with the associated design freedom of AM, this may provide the opportunity to not only enhance the efficiency of creating biomimetic models, but also to precisely control the biological output within such scaffolds.