Evaluation of hernia surgical meshes sterilised with ethylene oxide for adoption under UK regulations.

BACKGROUND: Low-cost meshes (LCM) have been successfully used in low-income countries (LIC) over the past decades, demonstrating comparable surgical outcomes to commercial meshes at a fraction of the cost. However, LIC sterilisation standards (autoclave sterilisation at 121 °C) do not meet UK regulations for medical devices, which require either ethylene oxide (EO) sterilisation or steam sterilisation at 134 °C. Therefore, the aim of this study was to sterilise UK LCM and characterise their mechanical properties and in vitro biocompatibility to verify whether EO sterilisation causes changes in the mechanical properties and biocompatibility of LCM. METHODS: EO sterilised LCM were used. Uniaxial tensile tests were performed to measure mechanical properties. Biocompatibility was measured through viability and morphology of Human Dermal Fibroblasts (HDFs) cultured in mesh-conditioned media, and by calculating the metabolic activity and proliferation of HDFs attached on the meshes, with alamarBlue assay. RESULTS: Break stress of LCM1 was significantly higher than LCM2 (p < 0.0001), while Young's modulus of LCM1 was significantly lower than LCM2 (p < 0.05) and there was no significant difference in break strain. Viability and morphology showed no significant difference between LCM and control. Attachment and proliferation of HDFs on LCM showed a better proliferation on LCM2 than LCM1, with values similar to the control at the final time point. CONCLUSIONS: We demonstrated that EO sterilisation affects LCM mechanical properties, but they still have values closer to the native tissues than the commercially available ones. We also showed that in vitro biocompatibility of LCM2 is not affected by EO sterilisation, as HDFs attached and proliferated on the mesh, while EO affected attachment on LCM1. A more detailed cost analysis of the potential savings for healthcare systems around the world needs to be performed to strengthen the cost-effectiveness of this frugal innovation.

Evaluating Oxygen Tensions Related to Bone Marrow and Matrix for MSC Differentiation in 2D and 3D Biomimetic Lamellar Scaffolds.

The physiological O2 microenvironment of mesenchymal stem cells (MSCs) and osteoblasts and the dimensionality of a substrate are known to be important in regulating cell phenotype and function. By providing the physiologically normoxic environments of bone marrow (5%) and matrix (12%), we assessed their potential to maintain stemness, induce osteogenic differentiation, and enhance the material properties in the micropatterned collagen/silk fibroin scaffolds that were produced in 2D or 3D. Expression of osterix (OSX) and vascular endothelial growth factor A (VEGFA) was significantly enhanced in the 3D scaffold in all oxygen environments. At 21% O2, OSX and VEGFA expressions in the 3D scaffold were respectively 13,200 and 270 times higher than those of the 2D scaffold. Markers for assessing stemness were significantly more pronounced on tissue culture polystyrene and 2D scaffold incubated at 5% O2. At 21% O2, we measured significant increases in ultimate tensile strength (p < 0.0001) and Young's modulus (p = 0.003) of the 3D scaffold compared to the 2D scaffold, whilst 5% O2 hindered the positive effect of cell seeding on tensile strength. In conclusion, we demonstrated that the 3D culture of MSCs in collagen/silk fibroin scaffolds provided biomimetic cues for bone progenitor cells toward differentiation and enhanced the tensile mechanical properties.

Creating Interactions between Tissue-Engineered Skeletal Muscle and the Peripheral Nervous System.

Effective models of mammalian tissues must allow and encourage physiologically (mimetic) correct interactions between co-cultured cell types in order to produce culture microenvironments as similar as possible to those that would normally occur in vivo. In the case of skeletal muscle, the development of such a culture model, integrating multiple relevant cell types within a biomimetic scaffold, would be of significant benefit for investigations into the development, functional performance, and pathophysiology of skeletal muscle tissue. Although some work has been published regarding the behaviour of in vitro muscle models co-cultured with organotypic slices of CNS tissue or with stem cell-derived neurospheres, little investigation has so far been made regarding the potential to maintain isolated motor neurons within a 3D biomimetic skeletal muscle culture platform. Here, we review the current state of the art for engineering neuromuscular contacts in vitro and provide original data detailing the development of a 3D collagen-based model for the co-culture of primary muscle cells and motor neurons. The devised culture system promotes increased myoblast differentiation, forming arrays of parallel, aligned myotubes on which areas of nerve-muscle contact can be detected by immunostaining for pre- and post-synaptic proteins. Quantitative RT-PCR results indicate that motor neuron presence has a positive effect on myotube maturation, suggesting neural incorporation influences muscle development and maturation in vitro. The importance of this work is discussed in relation to other published neuromuscular co-culture platforms along with possible future directions for the field.

Laminin promotes vascular network formation in 3D in vitro collagen scaffolds by regulating VEGF uptake.

Angiogenesis is an essential neovascularisation process, which if recapitulated in 3D in vitro, will provide better understanding of endothelial cell (EC) behaviour. Various cell types and growth factors are involved, with vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 key components. We were able to control the aggregation pattern of ECs in 3D collagen hydrogels, by varying the matrix composition and/or having a source of cells signalling angiogenic proteins. These aggregation patterns reflect the different developmental pathways that ECs take to form different sized tubular structures. Cultures with added laminin and thus increased expression of α6 integrin showed a significant increase (p<0.05) in VEGFR2 positive ECs and increased VEGF uptake. This resulted in the end-to-end network aggregation of ECs. In cultures without laminin and therefore low α6 integrin expression, VEGFR2 levels and VEGF uptake were significantly lower (p<0.05). These ECs formed contiguous sheets, analogous to the 'wrapping' pathway in development. We have identified a key linkage between integrin expression on ECs and their uptake of VEGF, regulated by VEGFR2, resulting in different aggregation patterns in 3D.

Effects of photochemical riboflavin-mediated crosslinks on the physical properties of collagen constructs and fibrils.

The use of collagen scaffold in tissue engineering is on the rise, as modifications to mechanical properties are becoming more effective in strengthening constructs whilst preserving the natural biocompatibility. The combined technique of plastic compression and cross-linking is known to increase the mechanical strength of the collagen construct. Here, a modified protocol for engineering these collagen constructs is used to bring together a plastic compression method, combined with controlled photochemical crosslinking using riboflavin as a photoinitiator. In order to ascertain the effects of the photochemical crosslinking approach and the impact of the crosslinks created upon the properties of the engineered collagen constructs, the constructs were characterized both at the macroscale and at the fibrillar level. The resulting constructs were found to have a 2.5 fold increase in their Young's modulus, reaching a value of 650 ± 73 kPa when compared to non-crosslinked control collagen constructs. This value is not yet comparable to that of native tendon, but it proves that combining a crosslinking methodology to collagen tissue engineering may offer a new approach to create stronger, biomimetic constructs. A notable outcome of crosslinking collagen with riboflavin is the collagen's greater affinity for water; it was demonstrated that riboflavin crosslinked collagen retains water for a longer period of time compared to non-cross-linked control samples. The affinity of the cross-linked collagen to water also resulted in an increase of individual collagen fibrils' cross-sectional area as function of the crosslinking. These changes in water affinity and fibril morphology induced by the process of crosslinking could indicate that the crosslinked chains created during the photochemical crosslinking process may act as intermolecular hydrophilic nanosprings. These intermolecular nanosprings would be responsible for a change in the fibril morphology to accommodate variable volume of water within the fibril.

The Isolation, Culture, and Cryopreservation of Human Rectus Sheath Fibroblasts.

The vast majority of pelvic and intra-abdominal surgery is undertaken through at least one incision, through either the linea alba or the rectus sheath. These connective tissue layers are formed from the aponeuroses of the rectus muscles (anterior and posterior rectus sheath) and are vital for the structural integrity of the abdominal wall. Poor healing of these connective tissues after surgery can lead to significant morbidity for patients, who can develop unsightly and painful incisional hernias. Fibroblasts within the rectus sheath are responsible for laying down and remodeling collagen during the healing process after surgery. Despite their importance for this healing process, such cells have not been studied in vitro. In order to carry out such work, researchers must first be able to isolate these cells from human tissue and culture them successfully so they may be used for experimentation. This article provides an extensive and detailed protocol for the isolation, culture, cryopreservation, and thawing of human rectus sheath fibroblasts (RSFs). In our hands, this protocol develops confluent cultures of primary fibroblasts within 2 weeks, and sufficient cultures ready for freezing and storage after a further 2 to 4 weeks. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Collagenase digestion of human rectus sheath and isolation of RSFs Alternate Protocol: Collagenase digestion of human rectus sheath and isolation of RSFs, digestion in flask Support Protocol: Cryopreservation and thawing of human RSFs.

Characterisation of human posterior rectus sheath reveals mechanical and structural anisotropy.

BACKGROUND: Our work aims to investigate the mechanical properties of the human posterior rectus sheath in terms of its ultimate tensile stress, stiffness, thickness and anisotropy. It also aims to assess the collagen fibre organisation of the posterior rectus sheath using Second-Harmonic Generation microscopy. METHODS: For mechanical analysis, twenty-five fresh-frozen samples of posterior rectus sheath were taken from six different cadaveric donors. They underwent uniaxial tensile stress testing until rupture either in the transverse (n = 15) or longitudinal (n = 10) plane. The thickness of each sample was also recorded using digital callipers. On a separate occasion, ten posterior rectus sheath samples and three anterior rectus sheath samples underwent microscopy and photography to assess collagen fibre organisation. FINDINGS: samples had a mean ultimate tensile stress of 7.7 MPa (SD 4.9) in the transverse plane and 1.2 MPa (SD 0.8) in the longitudinal plane (P < 0.01). The same samples had a mean Youngs modulus of 11.1 MPa (SD 5.0) in the transverse plane and 1.7 MPa (SD 1.3) in the longitudinal plane (P < 0.01). The mean thickness of the posterior rectus sheath was 0.51 mm (SD 0.13). Transversely aligned collagen fibres could be identified within the posterior sheath tissue using Second-Harmonic Generation microscopy. INTERPRETATION: The posterior rectus sheath displays mechanical and structural anisotropy with greater tensile stress and stiffness in the transverse plane compared to the longitudinal plane. The mean thickness of this layer is around 0.51 mm - consistent with other studies. The tissue is constructed of transversely aligned collagen fibres that are visible using Second-Harmonic Generation microscopy.

Spatiotemporal dynamics of re-innervation and hyperinnervation patterns by uninjured CGRP fibers in the rat foot sole epidermis after nerve injury.

The epidermis is innervated by fine nerve endings that are important in mediating nociceptive stimuli. However, their precise role in neuropathic pain is still controversial. Here, we have studied the role of epidermal peptidergic nociceptive fibers that are located adjacent to injured fibers in a rat model of neuropathic pain. Using the Spared Nerve Injury (SNI) model, which involves complete transections of the tibial and common peroneal nerve while sparing the sural and saphenous branches, mechanical hypersensitivity was induced of the uninjured lateral (sural) and medial (saphenous) area of the foot sole. At different time points, a complete foot sole biopsy was taken from the injured paw and processed for Calcitonin Gene-Related Peptide (CGRP) immunohistochemistry. Subsequently, a novel 2D-reconstruction model depicting the density of CGRP fibers was made to evaluate the course of denervation and re-innervation by uninjured CGRP fibers. The results show an increased density of uninjured CGRP-IR epidermal fibers on the lateral and medial side after a SNI procedure at 5 and 10 weeks. Furthermore, although in control animals the density of epidermal CGRP-IR fibers in the footpads was lower compared to the surrounding skin of the foot, 10 weeks after the SNI procedure, the initially denervated footpads displayed a hyper-innervation. These data support the idea that uninjured fibers may play a considerable role in development and maintenance of neuropathic pain and that it is important to take larger biopsies to test the relationship between innervation of injured and uninjured nerve areas.

A Systematic Review Examining the Experimental Methodology Behind In Vivo Testing of Hiatus Hernia and Diaphragmatic Hernia Mesh.

INTRODUCTION: Mesh implants are regularly used to help repair both hiatus hernias (HH) and diaphragmatic hernias (DH). In vivo studies are used to test not only mesh safety, but increasingly comparative efficacy. Our work examines the field of in vivo mesh testing for HH and DH models to establish current practices and standards. METHOD: This systematic review was registered with PROSPERO. Medline and Embase databases were searched for relevant in vivo studies. Forty-four articles were identified and underwent abstract review, where 22 were excluded. Four further studies were excluded after full-text review-leaving 18 to undergo data extraction. RESULTS: Of 18 studies identified, 9 used an in vivo HH model and 9 a DH model. Five studies undertook mechanical testing on tissue samples-all uniaxial in nature. Testing strip widths ranged from 1-20 mm (median 3 mm). Testing speeds varied from 1.5-60 mm/minute. Upon histology, the most commonly assessed structural and cellular factors were neovascularisation and macrophages respectively (n = 9 each). Structural analysis was mostly qualitative, where cellular analysis was equally likely to be quantitative. Eleven studies assessed adhesion formation, of which 8 used one of four scoring systems. Eight studies measured mesh shrinkage. DISCUSSION: In vivo studies assessing mesh for HH and DH repair are uncommon. Within this relatively young field, we encourage surgical and materials testing institutions to discuss its standardisation.

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects.

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

Neuregulin 1 Drives Morphological and Phenotypical Changes in C2C12 Myotubes: Towards De Novo Formation of Intrafusal Fibres In Vitro.

Muscle spindles are sensory organs that detect and mediate both static and dynamic muscle stretch and monitor muscle position, through a specialised cell population, termed intrafusal fibres. It is these fibres that provide a key contribution to proprioception and muscle spindle dysfunction is associated with multiple neuromuscular diseases, aging and nerve injuries. To date, there are few publications focussed on de novo generation and characterisation of intrafusal muscle fibres in vitro. To this end, current models of skeletal muscle focus on extrafusal fibres and lack an appreciation for the afferent functions of the muscle spindle. The goal of this study was to produce and define intrafusal bag and chain myotubes from differentiated C2C12 myoblasts, utilising the addition of the developmentally associated protein, Neuregulin 1 (Nrg-1). Intrafusal bag myotubes have a fusiform shape and were assigned using statistical morphological parameters. The model was further validated using immunofluorescent microscopy and western blot analysis, directed against an extensive list of putative intrafusal specific markers, as identified in vivo. The addition of Nrg-1 treatment resulted in a 5-fold increase in intrafusal bag myotubes (as assessed by morphology) and increased protein and gene expression of the intrafusal specific transcription factor, Egr3. Surprisingly, Nrg-1 treated myotubes had significantly reduced gene and protein expression of many intrafusal specific markers and showed no specificity towards intrafusal bag morphology. Another novel finding highlights a proliferative effect for Nrg-1 during the serum starvation-initiated differentiation phase, leading to increased nuclei counts, paired with less myotube area per myonuclei. Therefore, despite no clear collective evidence for specific intrafusal development, Nrg-1 treated myotubes share two inherent characteristics of intrafusal fibres, which contain increased satellite cell numbers and smaller myonuclear domains compared with their extrafusal neighbours. This research represents a minimalistic, monocellular C2C12 model for progression towards de novo intrafusal skeletal muscle generation, with the most extensive characterisation to date. Integration of intrafusal myotubes, characteristic of native, in vivo intrafusal skeletal muscle into future biomimetic tissue engineered models could provide platforms for developmental or disease state studies, pre-clinical screening, or clinical applications.

Guiding cell migration in 3D: a collagen matrix with graded directional stiffness.

While matrix stiffness has been implicated in cell adhesion and migration, most studies have focused on the effects of substrate stiffness in 2D. The present work describes a novel continuous stiffness gradient model for studying such processes in 3D. Wedge-shaped collagen scaffolds were compressed to produce sheets of a desired (0.1 mm) uniform thickness, but with increasing collagen density along the length of the sheet. Dynamic mechanical analysis, carried out on 1 mm wide strips obtained from the two ends and the middle of each sheet, showed that the elastic modulus increased from 1057 +/- 487 kPa to 2305 +/- 693 kPa at the soft and stiff end respectively and was 1835 +/- 31 kPa in the middle. In constructs seeded with agarose marker beads prior to compression, mean agarose bead density rose from 10 +/- 1 to 71 +/- 12 at the soft and stiff end respectively and was 19 +/- 5 in the middle, indicating successful engineering of a density gradient corresponding to the measured stiffness gradient. Growth-arrested human dermal fibroblasts, initially seeded evenly within such constructs, accumulated preferentially towards the stiff part of the gradient after 3 and 6 days in culture. Durotactic migration was significant after 6 days. This model provides a new means for studying cellular mechanotaxis and patterning cells which is controllable, biomimetic and in 3D.

The mechanobiology of tendon fibroblasts under static and uniaxial cyclic load in a 3D tissue engineered model mimicking native extracellular matrix.

Tendon mechanobiology plays a vital role in tendon repair and regeneration; however, this mechanism is currently poorly understood. We tested the role of different mechanical loads on extracellular matrix (ECM) remodelling gene expression and the morphology of tendon fibroblasts in collagen hydrogels, designed to mimic native tissue. Hydrogels were subjected to precise static or uniaxial loading patterns of known magnitudes and sampled to analyse gene expression of known mechano-responsive ECM-associated genes (Collagen I, Collagen III, Tenomodulin, and TGF-ß). Tendon fibroblast cytomechanics was studied under load by using a tension culture force monitor, with immunofluorescence and immunohistological staining used to examine cell morphology. Tendon fibroblasts subjected to cyclic load showed that endogenous matrix tension was maintained, with significant concomitant upregulation of ECM remodelling genes, Collagen I, Collagen III, Tenomodulin, and TGF-ß when compared with static load and control samples. These data indicate that tendon fibroblasts acutely adapt to the mechanical forces placed upon them, transmitting forces across the ECM without losing mechanical dynamism. This model demonstrates cell-material (ECM) interaction and remodelling in preclinical a platform, which can be used as a screening tool to understand tendon regeneration.

Generating intrafusal skeletal muscle fibres in vitro: Current state of the art and future challenges.

Intrafusal fibres are a specialised cell population in skeletal muscle, found within the muscle spindle. These fibres have a mechano-sensory capacity, forming part of the monosynaptic stretch-reflex arc, a key component responsible for proprioceptive function. Impairment of proprioception and associated dysfunction of the muscle spindle is linked with many neuromuscular diseases. Research to-date has largely been undertaken in vivo or using ex vivo preparations. These studies have provided a foundation for our understanding of muscle spindle physiology, however, the cellular and molecular mechanisms which underpin physiological changes are yet to be fully elucidated. Therefrom, the use of in vitro models has been proposed, whereby intrafusal fibres can be generated de novo. Although there has been progress, it is predominantly a developing and evolving area of research. This narrative review presents the current state of art in this area and proposes the direction of future work, with the aim of providing novel pre-clinical and clinical applications.

Identification of key factors in deep O2 cell perfusion for vascular tissue engineering.

Blood vessel engineering requires an understanding of the parameters governing the survival of resident vascular smooth muscle cells. We have developed an in vitro, collagen-based 3D model of vascular media to examine the correlation of cell density, O2 requirements, and viability. Dense collagen sheets (100 micron) seeded with porcine pulmonary artery smooth muscle cells (PASMCs) at low or high (11.6 or 23.2x10(6) cells/mL) densities were spiraled around a mandrel to create tubular constructs and cultured for up to 6 days in vitro, under both static and dynamic perfusion conditions. Real-time in situ monitoring showed that within 24 hours core O2 tension dropped from 140 mmHg to 20 mmHg and 80 mmHg for high and low cell density static cultures, respectively, with no significant cell death associated with the lowest O2 tension. A significant reduction in core O2 tension to 60 mmHg was achieved by increasing the O2 diffusion distance of low cell density constructs by 33% (p<0.05). After 6 days of static, high cell density culture, viability significantly decreased in the core (55%), with little effect at the surface (75%), whereas dynamic perfusion in a re-circulating bioreactor (1 ml/min) significantly improved core viability (70%, p<0.05), largely eliminating the problem. This study has identified key parameters dictating vascular smooth muscle cell behavior in 3D engineered tissue culture.

Transforming growth factor-beta1 stimulation enhances Dupuytren's fibroblast contraction in response to uniaxial mechanical load within a 3-dimensional collagen gel.

PURPOSE: A function of fibroblasts is the generation of cytomechanical force within their surrounding extracellular matrix. Abnormalities in force generation may be the cause of many pathologic conditions including scarring, and some fibroproliferative disorders such as Dupuytren's disease, which is the focus of this report. METHODS: This work investigated the cytomechanical responses of Dupuytren's-derived fibroblasts to externally applied mechanical force using a culture force monitor model, with and without stimulation with the fibrosis-linked cytokine, transforming growth factor-beta1 (TGF-beta1). We compared these responses with cytomechanical responses of fibroblasts derived from the transverse carpal ligament. RESULTS: Dupuytren's fibroblasts display a significantly greater ability to contract a collagen matrix compared with control fibroblasts, with a maximum generated force of 131 dynes (p < .001). These cells did not exhibit a characteristic plateau phase in the contraction, which indicates a delay in achieving tensional homeostasis from Dupuytren's-derived cells. After being subjected to uniaxial overload and underload, Dupuytren's fibroblasts responded by increased force generation, whereas control fibroblasts responded by a reduction in force in response to an overload, and contraction in response to an underload. These changes were exacerbated by the addition of the profibrotic factor TGF-beta1, with a significant increase in generated force for all cell types, in particular during the early phase of fibroblast attachment and contraction, and a positive contraction gradient in response to overloading forces. CONCLUSIONS: These data suggest that cells derived from this fibrotic disease display characteristic abnormalities in force generation profiles. Their default response to loading or underloading is contraction, or increased force generation. This work highlights the role of TGF-beta1 as a mechano-transduction cytokine, which has an influence on the early phase cell of force generation, as well as a role in mechanical responses of cells to external mechanical stimuli. This, in turn, may influence the progression of Dupuytren's disease and the high rates of recurrence seen postoperatively.

Engineering of a Functional Tendon Using Collagen As a Natural Polymer.

Reconstruction of a tendon rupture is surgically challenging as each end of the tendon retracts, leaving a substantial gap and direct repair is often not feasible. A tendon graft is required to bridge this defect and restore function. Presently, these gaps are filled with auto-, allo-, or synthetic grafts, but they all have clinical limitations. To address this issue, we developed tissue-engineered grafts by a rapid process using compressed type I collagen, which is the most dominant protein in the tendon. However, biomechanical properties were found to be unsuitable to withstand complete load-bearing in vivo. Hence, a modified suture technique was previously developed to reduce the load on the engineered collagen graft to aid integration in vivo. Using this technique, we tested engineered collagen grafts in vivo on a lapine model in three groups up to 12 weeks without immobilization. Gross observation at 3 and 12 weeks showed the bridge integrated without adhesions with a significant increase in the mechanical, structural and histological properties as compared to 1 week. Insertion of a tissue-engineered collagen graft using a novel load-bearing suture technique which partially loads in vivo showed integration, greater mechanical strength and no adhesion formation in the time period tested. This collagen graft has inherent advantages as compared to the present-day tendon grafts.

Hyaluronan derived nanoparticle for simvastatin delivery: evaluation of simvastatin induced myotoxicity in tissue engineered skeletal muscle.

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 myotubes 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.

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.

Modelling multi-scale cell-tissue interaction of tissue-engineered muscle constructs.

Expectation on engineered tissue substitute continues to grow, and for an effective development of a functional tissue and to control its quality, cellular mechanoresponse plays a key role. Although the mechanoresponse - in terms of cell-tissue interaction across scales - has been understood better in recent years, there are still technical limitations to quantitatively monitor the processes involved in the development of both native and engineered tissues. Computational (in silico) studies have been utilised to complement the experimental limitations and successfully applied to the prediction of tissue growth. We here review recent activities in the area of combined experimental and computational analyses of tissue growth, especially in the tissue engineering context, and highlight the advantages of such an approach for the future of the tissue engineering, using our own case study of predicting musculoskeletal tissue engineering construct development.