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To investigate if human oral mucosal fibroblasts (HOMF) from patients with limbal stem cell deficiency (LSCD) can be used as an autologous feeder layer to support the culture of epithelial cells for potential clinical use.HOMF were isolated from oral mucosal biopsies obtained from the following groups of patients with LSCD: aniridia, mucous membrane pemphigoid (MMP), Stevens-Johnson syndrome (SJS), and ectodermal dysplasia (ED). The ability of these cells to support the culture of human limbal epithelial cells (HLE) was compared to that of HOMF from non-LSCD donors and 3T3s commonly used to culture epithelial cells for use in the clinic to treat LSCD.HOMF were successfully obtained by explant culture for 3/3 aniridia patients, 3/3 MMP patients, 1/3 SJS patients, and 1/1 ED patients. All HOMF cultured from these LSCD groups supported the expansion of HLE with epithelial culture times and total colony forming efficiency (CFE) comparable to those achieved on HOMF isolated from donors without LSCD. PCR showed that all HLE cultured on LSCD donor HOMF expressed p63α, CK15, PAX6, CK12, and MUC16 as did HLE cultured on the control non-LSCD donor HOMF and 3T3s. Western blotting detected CK15 and MUC16 protein expression in all groups.HOMF from patients with LSCD can be successfully used to support the expansion of epithelial cells. These cells may therefore be useful as autologous feeder fibroblasts for the expansion of epithelial cells for use in the clinic to treat LSCD.

Background: Skeletal muscle extracellular vesicles (SM-EVs) regulate gene expression events in myogenic differentiation. Optimising effective SM-EV isolation methods offering high levels of purity will be important to accurately define their composition and functionality. Size-exclusion chromatography (SEC) applied in combination with ultrafiltration (UF) has the potential to increase sample throughput, scalability and selectivity. However, an optimal UF+SEC methodology has not been tested for the isolation of myotube derived EVs. Our aim was to compare two different UF protocols and define an optimal window of SEC fractions to maximise SM-EVs recovery and sample purity. Methods: C2C12 myotube conditioned medium was pre-concentrated using Amicon® Ultra 15 or Vivaspin®20, 100KDa UF columns and processed by SEC (IZON, qEV 70nm). The resulting thirty fractions obtained were individually analysed to identify an optimal fraction window for EV recovery. Results: EV markers Alix and TSG101 could be detected up to fraction 13, while CD9 and Annexin A2 only up to fraction 6. ApoA1+ lipoprotein contaminants were detected from fraction 6 onwards for both protocols. Amicon and Vivaspin UF preconcentration protocols led to qualitative and quantitative variations in EV marker profiles and purity. Eliminating lipoprotein co-isolation by reducing the SEC fraction window resulted in a net loss of particles, but increased measures of sample purity and had only a negligible impact on the presence of EV marker proteins. Conclusion: In conclusion, this study developed optimal UF+SEC protocols for the isolation of SM-EVs based on sample purity (fractions 1-5) and total abundance (fractions 2-10). The resulting protocols will be valuable in isolating highly pure SM-EV preparations for biomarker studies.

Skeletal muscle atrophy as a consequence of acute and chronic illness, immobilisation, muscular dystrophies and aging, leads to severe muscle weakness, inactivity and increased mortality. Mechanical loading is thought to be the primary driver for skeletal muscle hypertrophy, however the extent to which mechanical loading can offset muscle catabolism has not been thoroughly explored. In vitro 3D-models of skeletal muscle provide a controllable, high throughput environment and mitigating many of the ethical and methodological constraints present during in vivo experimentation. This work aimed to determine if mechanical loading would offset dexamethasone (DEX) induced skeletal muscle atrophy, in muscle engineered using the C2C12 murine cell line. Mechanical loading successfully offset myotube atrophy and functional degeneration associated with DEX regardless of whether the loading occurred before or after 24 h of DEX treatment. Furthermore, mechanical load prevented increases in MuRF-1 and MAFbx mRNA expression, critical regulators of muscle atrophy. Overall, we demonstrate the application of tissue engineered muscle to study skeletal muscle health and disease, offering great potential for future use to better understand treatment modalities for skeletal muscle atrophy. Electronic supplementary material The online version of this article (10.1007/s10974-020-09589-0) contains supplementary material, which is available to authorized users.

Extracellular vesicles (EVs) continue to gain interest across the scientific community for diagnostic and therapeutic applications. As EV applications diversify, it is essential that researchers are aware of challenges, in particular the compatibility of EV isolation methods with downstream applications and their clinical translation. We report outcomes of the first cross-comparison study looking to determine parameters (EV source, starting volume, operator experience, application and implementation parameters such as cost and scalability) governing the selection of popular EV isolation methods across disciplines. Our findings highlighted an increased clinical focus, with 36% of respondents applying EVs in therapeutics and diagnostics. Data indicated preferential selection of ultracentrifugation for therapeutic applications, precipitation reagents in clinical settings and size exclusion chromatography for diagnostic applications utilising biofluids. Method selection was influenced by operator experience, with increased method diversity when EV research was not the respondents primary focus. Application and implementation criteria were indicated to be major influencers in method selection, with UC and SEC chosen for their abilities to process large and small volumes, respectively. Overall, we identified parameters influencing method selection across the breadth of EV science, providing a valuable overview of practical considerations for the effective translation of research outcomes.

Background: Chronic Achilles and patellar tendinopathy are a significant burden in physically active populations. High-volume image-guided injection (HVIGI) proposes to strip away associated neovascularity, disrupt painful nerve ingrowth, and facilitate rehabilitation. Purpose: To investigate the efficacy of HVIGI with and without steroid relative to placebo. Study Design: Randomized controlled trial; Level of evidence, 1. Methods: A total of 62 participants were recruited between May 25, 2016, and March 5, 2020. Participants were men aged 18 to 55 years with Achilles or patellar tendinopathy of at least 6-month chronicity that had not improved with nonoperative management (including physical therapy and shockwave therapy), with ultrasound evidence of neovascularization, tendon thickening, and echogenic changes. They were assigned to the following groups: control (3 mL of subcutaneous 0.5% bupivacaine), HVIGI (10 mL of 0.5% bupivacaine and 30 mL of normal saline, ultrasound-guided between tendon and underlying fat pad), or HVIGI with steroid (HVIGIwSteroid; 0.25 mL of 100 mg/mL hydrocortisone). Clinicians and assessors were blinded. All participants were supervised through a pain-guided progressive loading program for 6 months postinjection. The main outcome measures were the Victoria Institute of Sport Assessments (VISA) for Achilles and patellar tendinopathy and the visual analog scale (VAS) for pain at 6 months postinjection. Results: The VISA score improved by a mean of 22.8 points (95% CI, 10.4-35.3 points; effect size [ES], 1.51) in the control group (n = 21), 18.6 points (95% CI, 9.1-28.0 points; ES, 1.31) in the HVIGI group (n = 21), and 18.5 points (95% CI, 3.4-33.6 points; ES, 0.88) in the HVIGIwSteroid group (n = 20). VAS pain improved by a mean of 15 points (interquartile range [IQR], –38.75, 8 points; ES, 0.39) in controls, 13 points (IQR,–34.0, 3.75 points; ES, 0.47) in the HVIGI group, and 27 points (IQR,–38.0, –1.0 points; ES, 0.54) in the HVIGIwSteroid group. The main effects were significant for time ( P < .001) but not group ( P ≥ .48), with no group × time interaction ( P = .71). One participant was lost to follow-up from each group, multiple imputation was used for missing data points. No adverse events occurred. Conclusion: Study findings did not demonstrate superiority of HVIGI over control injection. Registration: EU Clinical Trials Register (EudraCT: 2015-003587-36).

BackgroundChronic tendinopathy is a significant burden in physically active populations. High-volume image-guided injection (HVIGI) proposes to strip away associated neovascularity, disrupt painful nerve ingrowth and facilitate rehabilitation.MethodsThe objective was to investigate the efficacy of HVIGI with and without steroid relative to placebo. The study design was a three-arm block randomised control trial in a tertiary rehabilitation centre. Sixty-two participants were recruited between 25 May 2016 and 5 March 2020. Participants were men aged 18–55 with, Achilles or patellar tendinopathy of at least 6-months chronicity who had not improved with conservative management including shockwave, Ultrasound (US) evidence of neovascularisation, tendon thickening and echogenic changes.Participants were randomly assigned to control (3ml subcutaneous 0.5% Bupivacaine) or HVIGI (10 ml 0.5% Bupivacaine and 30 ml normal saline US guided between tendon and underlying fatpad) or HVIGI with steroid (0.25 ml 100 mg/ml hydrocortisone). Clinicians and assessors were blinded. All participants were supervised through a pain-guided progressive loading programme for 6-months post injection. The primary outcome measures were the Victoria institute of sport assessments for Achilles and patellar tendinopathy (VISA-A and VISA-P) and visual analogue scale (VAS)-pain at 6-months.ResultsVISA improved by 22.8 ((95% CI) 10.4 to 35.3, effect size (ES) 1.51) in control (n=21), 18.6 (9.1 to 28.0, ES 1.31) in HVIGI (n=21) and 18.5 (3.4 to 33.6, ES 0.88) in HVIGI with steroid (n=20) groups. VAS-pain improved by 15 ((IQR) -38.75 to 8, ES 0.39) in control, 13 in HVIGI (-34.0 to 3.75, ES 0.47) and 27 (-38.0 to-1.0, ES 0.54) in HVIGI with steroid groups. Main effects for time were significant (pConclusionsData does not demonstrate superiority of HVIGI over control injection. Pain-guided progressive loading remains the mainstay of treatment.

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.

Cellular metabolism and behaviour is closely linked to cytoskeletal tension and scaffold mechanics. In the developing nervous system functional connectivity is controlled by the interplay between chemical and mechanical cues that initiate programs of cell behaviour. Replication of functional connectivity in neuronal populations in vitro has proven a technical challenge due to the absence of many systems of biomechanical regulation that control directional outgrowth in vivo. Here, a 3D culture system is explored by dilution of a type I collagen hydrogel to produce variation in gel stiffness. Hydrogel scaffold remodelling was found to be linked to gel collagen concentration, with a greater degree of gel contraction occurring at lower concentrations. Gel mechanics were found to evolve over the culture period according to collagen concentration. Less concentrated gels reduced in stiffness, whilst a biphasic pattern of increasing and then decreasing stiffness was observed at higher concentrations. Analysis of these cultures by PCR revealed a program of shifting integrin expression and highly variable activity in key morphogenic signal pathways, such as mitogen-associated protein kinase, indicating genetic impact of biomaterial interactions via mechano-regulation. Gel contraction at lower concentrations was also found to be accompanied by an increase in average collagen fibre diameter. Minor changes in biomaterial mechanics result in significant changes in programmed cell behaviour, resulting in adoption of markedly different cell morphologies and ability to remodel the scaffold. Advanced understanding of cell-biomaterial interactions, over short and long-term culture, is of critical importance in the development of novel tissue engineering strategies for the fabrication of biomimetic 3D neuro-tissue constructs. Simple methods of tailoring the initial mechanical environment presented to SH-SY5Y populations in 3D can lead to significantly different programs of network development over time.

Biodegradable magnesium (Mg) has garnered attention for its use in orthopaedic implants due to mechanical properties that closely match to those of bone. Studies have been undertaken to understand the corrosion behaviour of these materials and their effects on bone forming cells. However, there is lack of research on how the corrosion of these biomaterials affect surrounding tissues such as skeletal muscle. Mg plays an important role in the structural and functional properties of skeletal muscle. It is therefore important to investigate the response of skeletal muscle cells to both soluble (Mg ions) and insoluble (corrosion granules) corrosion products. Through in vitro studies it is possible to observe the effects of corrosion products on myotube formation by the fusion of single muscle precursor cells known as myoblasts. To achieve this goal, it is important to determine if these corrosion products are toxic to myotubes. Here it was noted that although there was a slight decrement in cellular viability after initial exposure, this soon recovered to control levels. A high Ca/Mg ratio resulted in the formation of large myotubes and a low Ca/Mg ratio negatively affected myotube maturation. Mg2+ and Ca2+ ions are important in the process of myogenesis, and the concentration of these ions and the ratio of the ions to each other played a significant role in myotube cellular activity. The outcomes of this study could pave the way to a bio-informed and integrated approach to the design and engineering of Mg-based orthopaedic implants.

Three-dimensional tissue-engineered structures enable more representative determination of novel drug or material effects on tissue than traditional monolayer cell cultures. This study sought to better understand how key manufacturing variables affect the myotube characteristics of a skeletal muscle model toward reducing resource use and to develop an understanding of scaling on model consistency. C2C12 murine myoblasts were seeded in a tethered collagen scaffold from which directional myotubes form in response to lines of tension and a change in medium. Collagen polymerizing area length-to-width ratios greater than one were found to reduced cell-matrix attachment and remodeling forces significantly (p

Knee valgus alignment has been associated with lower-limb musculoskeletal injury. This case-control study aims to: assess biomechanical differences between patients with patellar tendinopathy and healthy controls.43 military participants (21 cases, 22 controls) were recorded using 3D-motion capture performing progressively demanding, small knee bend, single leg and single leg decline squats. Planned a priori analysis of peak: hip adduction, knee flexion, pelvic tilt, pelvic obliquity and trunk flexion was conducted using MANOVA. Kinematic and kinetic data were graphed with bootstrapped t-tests and 95% CI's normalised to the squat cycle. ANOVA and correlations in SPSS were used for exploratory analysis.On their symptomatic side cases squatted to less depth (-6.62°, p 0.05) than controls with exploratory curve analysis revealing a pattern of increased knee valgus collapse throughout the squatting movement (p 0.05). Greater patella tendon force was generated by: the eccentric than concentric phase of squatting (+30-43%, ES 0.52-1.32, p 0.01), declined (plantarflexed) compared to horizontal surface (+36-51%, ES 1.19-1.68, p 0.01) and deeper knee flexion angles (F ≥ 658.3, p 0.01) with no difference between groups (F ≤ 1.380, p 0.05). Cases experienced more pain on testing on decline board (ES = 0.69, p 0.01). For symptomatic limbs pain (rKnee valgus alignment is a plausible risk factor for patellar tendinopathy. Conclusions relating to causation are limited by the cross-sectional study design. Increasing squat depth, use of a declined surface and isolating the eccentric phase enable progression of loading prescription guided by pain.

The need for self-directed learning for professional development drives an increase in the delivery of easy to use {\textquoteleft}just-in-time{\textquoteright} resources that respond to the often-dynamic workplace and work culture. This is especially important in the era of globalisation, when the number of employees, who are culturally diverse, increases each year. Most medium and large companies operate in an international environment, and this is due to the expansion of international enterprises with branches in various countries that requires cooperation with foreign clients, and the employment of foreign nationals in their companies. In order to guarantee the effectiveness of workings in companies, there is a need for continuous education in the aspect of the cultural diversity. This paper explores micro-learning, which focuses on delivering brevity through bite-sized learning units or short-term learning activities. Learning content in this case can take many forms, from text to interactive multimedia. These contents are often created on demand, which can sometimes be less contextualised and pedagogically informed. Based on a case study of the need for training on cultural risks in multi-cultural organisations, this paper focuses on the design of mini-games as playful learning resources for supporting an online learning platform that has been developed as a response to this training need. Fifteen mini-games have been developed to complement eight main topics related to cultural risks and to promote reflection, practice and the self-assessment of knowledge acquired through the platform. The main eight topics represent the risk areas identified that include cultural awareness, understanding different cultures, communication, learning styles, hierarchy, team-working, qualities in the working place, and stereotypes through a survey carried out with personnel (n=154) from multi-cultural organisations across five countries - Cyprus, Italy, Latvia, Poland, and the UK. The discussions include unpacking the mapping of pedagogical and gameful design considerations based on Arnab et al.{\textquoteleft}s (2015) Learning Mechanics-Game Mechanics Mapping (LMGM) model. The paper also discusses the findings from the testing of the online platform across 5 countries including 166 participants (two-step testing). The insights provided will be valuable to researchers, practitioners, designers, and developers of micro-learning resources.

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.

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-1β 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.

The disordered environment found in conventional neural cultures impedes various applications where cell directionality is a key process for functionality. Neurons are highly specialized cells known to be greatly dependent on interactions with their surroundings. Therefore, when chemical cues are incorporated on the surface material, a precise control over neuronal behavior can be achieved. Here, the behavior of SH-SY5Y neurons on a variety of self-assembled monolayers (SAMs) and polymer brushes both in isolation and combination to promote cellular spatial control was determined. APTES and BIBB coatings promoted the highest cell viability, proliferation, metabolic activity, and neuronal maturation, while low cell survival was seen on PKSPMA and PMETAC surfaces. These cell-attractive and repulsive surfaces were combined to generate a binary BIBB-PKSPMA coating, whereby cellular growth was restricted to an exclusive neural region. The utility of these coatings when precisely combined could act as a bioactive/bioinert surface resulting in a biomimetic environment where control over neuronal growth and directionality can be achieved.

Zebrafish (Danio rerio) are a commonly used model organism to study human muscular myopathies and dystrophies. To date, much of the work has been conductedin vivodue to limitations surrounding the consistent isolation and culture of zebrafish muscle progenitor cells (MPCs)in vitroand the lack of physiologically relevant models.Here we report a robust, repeatable, and cost-effective protocol for the isolation and culture of zebrafish MPCs in conventional monolayer (2D) and have successfully transferred these cells to 3D culture in collagen based three-dimensional (3D) tissue-engineered constructs. Zebrafish MPC’s cultured in 2D were consistently reported to be Desmin positive reflecting their muscle specificity, with those demonstrating Desmin positivity in the 3D cultures. In addition, mRNA expression of muscle markers specific for proliferation, differentiation and maturation measured from both monolayer and 3D cultures at appropriate developmental stages were found consistent with previously published from other speciesin vitro and in vivomuscle data.Collagen constructs seeded with zebrafish MPC’s were initially characterised for optimal seeding density, followed by macroscopic characterisation (three-fold contraction) of the matrix. Direct comparison between the morphological characteristics (proportion of cells) and gene expression profiles of cells cultured in collagen constructs revealed higher maturation and differentiation compared to monolayer cultures. In this regard, cells embedded in 3D collagen constructs revealed higher fusion index, Desmin positivity, hypertrophic growth, myotube maturity and myogenic mRNA expression when compared to in monolayer.In conclusion, these methods and models developed herein will facilitatein vitroexperiments, which would complementin vivozebrafish studies used to investigate the basic developmental, myopathies and dystrophies in skeletal muscle cells.

BackgroundSkeletal muscle (SkM) regenerates following injury, replacing damaged tissue with high fidelity. However, in serious injuries, non-regenerative defects leave patients with loss of function, increased re-injury risk and often chronic pain. Progress in treating these non-regenerative defects has been slow, with advances only occurring where a comprehensive understanding of regeneration has been gained. Tissue engineering has allowed the development of bioengineered models of SkM which regenerate following injury to support research in regenerative physiology. To date, however, no studies have utilised human myogenic precursor cells (hMPCs) to closely mimic functional human regenerative physiology.ResultsHere we address some of the difficulties associated with cell number and hMPC mitogenicity using magnetic association cell sorting (MACS), for the marker CD56, and media supplementation with fibroblast growth factor 2 (FGF-2) and B-27 supplement. Cell sorting allowed extended expansion of myogenic cells and supplementation was shown to improve myogenesis within engineered tissues and force generation at maturity. In addition, these engineered human SkM regenerated following barium chloride (BaCl2) injury. Following injury, reductions in function (87.5%) and myotube number (33.3%) were observed, followed by a proliferative phase with increased MyoD+ cells and a subsequent recovery of function and myotube number. An expansion of the Pax7+ cell population was observed across recovery suggesting an ability to generate Pax7+ cells within the tissue, similar to the self-renewal of satellite cells seen in vivo.ConclusionsThis work outlines an engineered human SkM capable of functional regeneration following injury, built upon an open source system adding to the pre-clinical testing toolbox to improve the understanding of basic regenerative physiology.

Herein, the cytotoxicity of a novel zwitterionic sulfobetaine hydrogel system with a nano-clay crosslinker has been investigated. We demonstrate that careful selection of the composition of the system (monomer to Laponite content) allows the material to be formed into controlled shapes using an extrusion based additive manufacturing technique with the ability to tune the mechanical properties of the product. Moreover, the printed structures can support their own weight without requiring curing during printing which enables the use of a printing-then-curing approach. Cell culture experiments were conducted to evaluate the neural cytotoxicity of the developed hydrogel system. Cytotoxicity evaluations were conducted on three different conditions; a control condition, an indirect condition (where the culture medium used had been in contact with the hydrogel to investigate leaching) and a direct condition (cells growing directly on the hydrogel). The result showed no significant difference in cell viability between the different conditions and cells were also found to be growing on the hydrogel surface with extended neurites present.

Skeletal muscle (SkM) regenerates following injury, replacing damaged tissue with high fidelity. However, in serious injuries non-regenerative defects leave patients with loss of function, increased re-injury risk and often chronic pain. Progress in treating these non-regenerative defects has been slow, with advances only occurring where a comprehensive understanding of regeneration has been gained. Tissue engineering has allowed the development of bioengineered models of SkM which regenerate following injury to support research in regenerative physiology. To date however, no studies have utilised human myogenic precursor cells (hMPCs) to closely mimic human physiology due to difficulties generating sufficient cell numbers and the relatively low myogenic potential of hMPCs. Here we address problems associated with cell number and hMPC mitogenicity using magnetic association cell sorting (MACS), for the marker CD56, and media supplementation with fibroblast growth factor 2 (FGF-2) and B-27 supplement. Cell sorting allowed extended expansion of myogenic cells and supplementation was shown to improve myogenesis within engineered tissues and force generation at maturity. In addition, these engineered human SkM contained a Pax7+ niche and regenerated following Barium Chloride (BaCl2) injury. Following injury, reductions in function (87.5%) and myotube number (33.3%) were observed, followed by a proliferative phase with increased MyoD+ cells and a subsequent recovery of function and myotube number. An expansion of the Pax7+ cell population was observed across recovery suggesting an ability to generate Pax7+ cells within the tissue, similar to the self-renewal of satellite cells seen in vivo. This work outlines an engineered human SkM capable of functional regeneration following injury, built upon an open source system adding to the pre-clinical testing toolbox to improve the understanding of basic regenerative physiology.

Statins are currently the most prescribed hypercholesterolemia-lowering drugs worldwide, with estimated usage approaching one-sixth of the population. However, statins are known to cause pleiotropic skeletal myopathies in 1.5% to 10% of patients and the mechanisms by which statins induce this response, are not fully understood. In this study, a 3D collagen-based tissue-engineered skeletal muscle construct is utilised as a screening platform to test the efficacy and toxicity of a new delivery system. A hyaluronic acid derived nanoparticle loaded with simvastatin (HA-SIM-NPs) is designed and the effect of free simvastatin and HA-SIM-NPs on cellular, molecular and tissue response is investigated. Morphological ablation of myo-tubes and lack of de novo myotube formation (regeneration) was evident at the highest concentrations (333.33 µM), independent of delivery vehicle (SIM or HA-SIM-NP). A dose-dependent disruption of the cytoskeleton, reductions in metabolic activity and tissue engineered (TE) construct tissue relaxation was evident in the free drug condition (SIM, 3.33 µM and 33.33 nM). However, most of these changes were ameliorated when SIM was delivered via HA-SIM-NPs. Significantly, homogeneous expressions of MMP2, MMP9, and myogenin in HA-SIM-NPs outlined enhanced regenerative responses compared to SIM. Together, these results outline statin delivery via HA-SIM-NP as an effective delivery mechanism to inhibit deleterious myotoxic side-effects. publishedVersion

3D printing is becoming increasingly prevalent in modern chemistry laboratories. This technology provides chemists with the ability to design, prototype and print functional devices that integrate catalytic and/or analytical functionalities and even to print common laboratory hardware and teaching aids. Although access to 3D printers has increased considerably, some design principles and material considerations need to be weighed before employing such technology in chemistry laboratories. In addition, a certain level of expertise needs to be acquired in order to use computer-aided design, printing software and the specialist hardware associated with higher-end instrumentation. Nonetheless, the recent progress in this field is encouraging, with these printing technologies offering many advantages over traditional production methods. This Review highlights some of the notable advances in this growing area over the past decade. 3D printing is becoming a mainstream technology with considerable increase in access to affordable desktop printers. However, specific design principles and material considerations need to be weighed when printing functional devices that integrate catalytic and/or analytical functionalities, as well as when printing common laboratory hardware.

Hyperinsulinaemia potentially contributes to insulin resistance in metabolic tissues, such as skeletal muscle. The purpose of these experiments was to characterise glucose uptake, insulin signalling and relevant gene expression in primary human skeletal muscle-derived cells (HMDCs), in response to prolonged insulin exposure (PIE) as a model of hyperinsulinaemia-induced insulin resistance. Differentiated HMDCs from healthy human donors were cultured with or without insulin (100 nM) for 3 days followed by an acute insulin stimulation. HMDCs exposed to PIE were characterised by impaired insulin-stimulated glucose uptake, blunted IRS-1 phosphorylation (Tyr612) and Akt (Ser473) phosphorylation in response to an acute insulin stimulation. Glucose transporter 1 (GLUT1), but not GLUT4, mRNA and protein increased following PIE. The mRNA expression of metabolic (PDK4) and inflammatory markers (TNF-α) was reduced by PIE but did not change lipid (SREBP1 and CD36) or mitochondrial (UCP3) markers. These experiments provide further characterisation of the effects of PIE as a model of hyperinsulinaemia-induced insulin resistance in HMDCs.

Resolution of inflammation is now known to be an active process which in part is instigated and controlled by specialized pro-resolving lipid mediators (SPM's) derived from dietary omega-3 fatty acids. Resolvin E1 (Rv E1 ) is one of these SPM's derived from the omega-3 fatty acid eicosapentaenoic acid. Using both molecular and phenotypic functional measures we report that in a model of Lipopolysaccharide (LPS) induced inflammation, Rv E1 attenuated mRNA levels of both interlukin-6 and monocyte chemoattractant protein-1 whilst having no effect on tumor necrosis factor-α or interlukin-1β in C2C12 skeletal muscle myotubes. Findings at the molecular level were transferred into similar changes in extracellular protein levels of the corresponding genes with the greatest attenuation being noted in IL-6 protein concentrations. Rv E1 instigated beneficial morphological changes through the prevention of LPS induced skeletal muscle atrophy, in tandem with attenuation of the LPS induced reduction in contractile force in tissue engineered skeletal muscle. These findings demonstrate, in our model of endotoxin induced inflammation in skeletal muscle, that Rv E1 has pro-resolving properties in this cell type. Our data provides rationale for further investigation into the mechanistic action of Rv E1 in skeletal muscle, with the vision of having potential benefits for the prevention/resolution of in-vivo skeletal muscle atrophy.

Background Single leg squats are commonly used subjective assessments of general biomechanical function, injury risk, as a predictor for recovery and as an outcome measure of rehabilitation. While 3D motion capture is a useful tool for elite sports performance and research it is impractical for routine clinical use. Research question This cross-sectional study aims to: assess reliability and validity of clinicians’ subjective ratings of single leg squats compared to 3D motion capture, and to identify whether performance predicts joint moments. Methods 22 healthy military volunteers were simultaneously recorded on video and 3D motion capture performing single leg squats. Videos were reviewed twice by 5 physiotherapists rating performance on a 0–5 scale assessing squat depth, hip adduction, pelvic obliquity, pelvic tilt and trunk flexion summated into a composite score. Results Hip adduction and trunk flexion exhibited moderate to substantial inter- and intra-rater reliability (range κ = 0.408–0.699) other individual criteria were mostly fair (κ ≤ 0.4). Composite scores for inter-rater reliability were ICC (1,1) = 0.419 and ICC (1,κ) = 0.783 and intra-rater reliability were ICC (1,1) = 0.672 and κ(w) = 0.526. Validity against 3D kinematics was poor with only 6/75 individually rated criteria reaching κ > 0.40. Correlation was found between composite scores and hip internal rotation moment (r s = 0.571, p = 0.009). Significance Repeated use of single leg squats by a single practitioner is supported. Comparisons between clinicians are unreliable but improved by average measures from multiple raters. Heterogeneous reliability across scoring components suggests a qualitative description of the criteria scored is less ambiguous than using composite scores in a clinical setting. Composite scores may be more useful for analysis at a population level. Poor validity against kinematic data suggests clinicians use additional information upon which they find agreement such as estimating kinetics. Correlation between hip internal rotation moment and subjective ratings may be such an example of clinicians trying to identify excessive abnormal loading.

Contemporary tissue engineered skeletal muscle models display a high degree of physiological accuracy compared with native tissue, and therefore may be excellent platforms to understand how various pathologies affect skeletal muscle. Chronic obstructive pulmonary disease (COPD) is a lung disease which causes tissue hypoxia and is characterized by muscle fiber atrophy and impaired muscle function. In the present study we exposed engineered skeletal muscle to varying levels of oxygen (O2 ; 21-1%) for 24 h in order to see if a COPD like muscle phenotype could be recreated in vitro, and if so, at what degree of hypoxia this occurred. Maximal contractile force was attenuated in hypoxia compared to 21% O2 ; with culture at 5% and 1% O2 causing the most pronounced effects with 62% and 56% decrements in force, respectively. Furthermore at these levels of O2 , myotubes within the engineered muscles displayed significant atrophy which was not seen at higher O2 levels. At the molecular level we observed increases in mRNA expression of MuRF-1 only at 1% O2 whereas MAFbx expression was elevated at 10%, 5%, and 1% O2 . In addition, p70S6 kinase phosphorylation (a downstream effector of mTORC1) was reduced when engineered muscle was cultured at 1% O2 , with no significant changes seen above this O2 level. Overall, these data suggest that engineered muscle exposed to O2 levels of ≤5% adapts in a manner similar to that seen in COPD patients, and thus may provide a novel model for further understanding muscle wasting associated with tissue hypoxia. J. Cell. Biochem. 118: 2599-2605, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

Heterotopic ossification (HO) is a debilitating condition defined by the rapid formation of bone in soft tissues. What makes HO fascinating is firstly the rate at which bone is deposited, and secondly the fact that this bone is structurally and compositionally similar to that of a healthy adult. If the mechanisms governing HO are understood, they have the potential to be exploited for the development of potent osteoinductive therapies. With this aim, we utilised a tissue engineered skeletal muscle model to better understand the role of inflammation on this debilitating phenomenon. We showed myoblasts could be divided into two distinct populations, myogenic cells and undifferentiated "reserve" cells. Gene expression analysis of myogenic and osteo-regulatory markers confirmed that "reserve" cells were primed for osteogenic differentiation, but had a reduced capacity for myogenesis. Osteogenic differentiation was significantly enhanced in the presence of PDGF-BB and BMP2, and correlated with conversion to a Sca-1(+) /CD73(+) phenotype. Alizarin red staining showed that PDGF-BB promoted significantly more mineral deposition than BMP2. Finally, we showed that PDGF-induced mineralisation was blocked in the presence of the pro-inflammatory cytokines TNFα and IL1. In conclusion, the present study identified that PDGF-BB is a potent osteoinductive factor in a model of tissue engineered skeletal muscle, and that the osteogenic capacity of this protein was modulated in the presence of pro-inflammatory cytokines. These findings reveal a possible mechanism by which HO develops following trauma. Importantly, these findings have implications for the induction and control of bone formation for regenerative medicine.

To improve the biological performance of hydroxyapatite scaffolds in bone tissue engineering, graphite was used as porogen to create additional micro/nanoporosity to macroporosity, resulting in hierarchical porosity. For maximum imitation of natural bone structures, scaffolds with different porosity features were fabricated using micron/nano-sized graphite. The sintering profile of graphite treated scaffolds was optimised to reduce the influence of shrinkage. To confirm the porosity features, the micro/nanostructures of scaffolds were characterised by scanning electron microscopy and Brunauer-Emmett-Teller method. Considering that hydroxyapatite is resistant to biodegradation in vivo, the degradation rate of scaffolds in modified simulated body fluid was examined. Furthermore, biological evaluations based on myoblasts were carried out to investigate the influence of porosity features on the essential performance such as adhesion, proliferation and differentiation. The results indicate that the scaffolds with dominant microporosity and little nanoporosity formed inside had high potential for clinical applications due to improved performance in bioactivity.

Cultured limbal and oral epithelial cells have been successfully used to treat patients with limbal stem cell deficiency (LSCD). The most common culture method for these cell therapies utilizes amniotic membrane as a cell support and/or murine 3T3s as feeder fibroblasts. The aim of this study is to refine the production of autologous oral mucosal cell therapy for the treatment of LSCD. Real architecture for 3D tissue (RAFT) is used as an alternative cell culture support. In addition, oral mucosal cells (epithelial and fibroblast) are used as autologous alternatives to donor human limbal epithelial cells (HLE) and murine 3T3s. The following tissue equivalents are produced and characterized: first, for patients with bilateral LSCD, an oral mucosa tissue equivalent consisting of human oral mucosal epithelial cells on RAFT supported by human oral mucosal fibroblasts (HOMF). Second, for patients with unilateral LSCD, HLE on RAFT supported by HOMF. For both tissue equivalent types, features of the cornea are observed including a multi-layered epithelium with small cells with a stem cell like phenotype in the basal layer and squamous cells in the top layers, and p63α and PAX6 expression. These tissue equivalents may therefore be useful in the treatment of LSCD.