No abstract

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.

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 (C C ), 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. C C , 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.

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 CC 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 CC 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. CC 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.

Background 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 functional human regenerative physiology. Results Here 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. Conclusions 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.