Skeletal Muscle Tissue Engineering Research Articles

Global Evolution of Skeletal Muscle Tissue Engineering: A Scientometric Research.

Skeletal muscle tissue engineering (SMTE) is of great significance in the study of skeletal muscle physiology and pathology, which could be used in skeletal muscle graft. The scientometric analysis of SMTE can help researchers to quickly understand the evolutive history, status, novelties, and trend of this field. In this study, we performed a scientometric study that can be used to construct and visualize networks of SMTE using VOSviewer. A total of 1384 documents published between 1994 and 2020 were retrieved and analyzed. Our results showed that number of publications in SMTE has increased slowly from 1994 to 2014 and has increased rapidly from 2015 to 2020. The geographical distribution of publications in terms of total publications about SMTE is concentrated in Europe and the United States. The most productive institution was University of Michigan, while Harvard University and the University of Pittsburgh were ranked the second and third places. SMTE influenced a wide spectrum of disciplines, including Biology and Medicine and Physical Sciences. In addition, the research hotspot of SMTE was expanding from seed cells to the combination with advanced strategies (electrostatic spinning, bioprinting, and materials) for emulating the highly bionic engineered skeletal muscle tissues. This study provided a unique perspective for understanding the history and trends of SMTE, which could help to promote the rapid development of the field. Impact statement Skeletal muscle tissue engineering (SMTE), which acts as an important branch of tissue engineering, hold a great promise in the study of skeletal muscle physiology and pathology. The field of SMTE has developed rapidly in recent decades while still lacking studies based on scientometric methods. This article provided the first scientometric study of SMTE from development trends and evolution of the field. The results indicated that the field of SMTE was experiencing rapid growth and had a significant impact on multiple fields, particularly in Biology and Medicine and Physical Sciences.

Myogenic Potential of Extracellular Matrix Derived from Decellularized Bovine Pericardium.

Skeletal muscles represent 40% of body mass and its native regenerative capacity can be permanently lost after a traumatic injury, congenital diseases, or tumor ablation. The absence of physiological regeneration can hinder muscle repair preventing normal muscle tissue functions. To date, tissue engineering (TE) represents one promising option for treating muscle injuries and wasting. In particular, hydrogels derived from the decellularized extracellular matrix (dECM) are widely investigated in tissue engineering applications thanks to their essential role in guiding muscle regeneration. In this work, the myogenic potential of dECM substrate, obtained from decellularized bovine pericardium (Tissuegraft Srl), was evaluated in vitro using C2C12 murine muscle cells. To assess myotubes formation, the width, length, and fusion indexes were measured during the differentiation time course. Additionally, the ability of dECM to support myogenesis was assessed by measuring the expression of specific myogenic markers: α-smooth muscle actin (α-sma), myogenin, and myosin heavy chain (MHC). The results obtained suggest that the dECM niche was able to support and enhance the myogenic potential of C2C12 cells in comparison of those grown on a plastic standard surface. Thus, the use of extracellular matrix proteins, as biomaterial supports, could represent a promising therapeutic strategy for skeletal muscle tissue engineering.

Adipogenic Differentiation Alters Properties of Vascularized Tissue-Engineered Skeletal Muscle.

Advances in the engineering of comprehensive skeletal muscle models in vitro will improve drug screening platforms and can lead to better therapeutic approaches for the treatment of skeletal muscle injuries. To this end, a vascularized tissue-engineered skeletal muscle (TE-SkM) model that includes adipocytes was developed to better emulate the intramuscular adipose tissue that is observed in skeletal muscles of patients with diseases such as diabetes. Muscle precursor cells cultured with and without microvessels derived from adipose tissue (microvascular fragments) were used to generate TE-SkM constructs, with and without a microvasculature, respectively. TE-SkM constructs were treated with adipogenic induction media to induce varying levels of adipogenesis. With a delayed addition of induction media to allow for angiogenesis, a robust microvasculature in conjunction with an increased content of adipocytes was achieved. The augmentation of vascularized TE-SkM constructs with adipocytes caused a reduction in maturation (compaction), mechanical integrity (Young's modulus), and myotube and vessel alignment. An increase in basal glucose uptake was observed in both levels of adipogenic induction, and a diminished insulin-stimulated glucose uptake was associated with the higher level of adipogenic differentiation and the greater number of adipocytes.

Regulation of Myogenic Differentiation by Topologically Microgrooved Surfaces for Skeletal Muscle Tissue Engineering.

Inspired by the natural topological structure of skeletal muscle tissue, the topological surface construction of bionic scaffolds for skeletal muscle repair has attracted great interest. Many previous studies have focused on the effects of the topological structure on myoblasts. However, these studies used only specific repeating sizes and shapes to achieve the myoblast alignment and myotube formation; moreover, the regulatory effects of the size of a topological structure on myogenic differentiation are often neglected, leading to a lack of guidance for the design of scaffolds for skeletal muscle tissue engineering. In this study, we fabricated a series of microgroove topographies with various widths and depths via a combination of soft lithography and melt-casting and studied their effects on the behaviors of skeletal muscle cells, especially myogenic differentiation, in detail. Microgrooved poly(lactic-co-glycolic acid) substrates were found to effectively regulate the proliferation, myogenic differentiation, and myotube formation of C2C12 cells, and the degree of myogenic differentiation was significantly dependent on signals in response to the size of the microgroove structure. Compared with their depth, the width of the microgroove structures can more strongly affect the myogenic differentiation of C2C12 cells, and the degree of myoblast differentiation was enhanced with increasing groove width. Microgroove structures with relatively large groove widths and small groove depths promoted the myogenic differentiation of C2C12 cells. In addition, the integrin-mediated focal adhesion kinase signaling pathway and MAPK signaling pathway were activated in cells in response to the external topological structure, and the size of the topological structure of the material surface effectively regulated the degree of the cellular response to the external topological structure. These results can guide the design of scaffolds for skeletal muscle tissue engineering and the construction of effective bionic scaffold surfaces for skeletal muscle regeneration.

Electrohydrodynamic-direct-printed cell-laden microfibrous structure using alginate-based bioink for effective myotube formation

In this study, a fully aligned microfibrous structure fabricated using fibrin-assisted alginate bioink and electrohydrodynamic direct-printing was proposed for skeletal muscle tissue engineering. To safely construct the aligned alginate/fibrin microfibrous structure laden with myoblasts or endothelial cells, various printing conditions, such as an applied electric field, distance between the nozzle and target, and nozzle moving speed, were selected appropriately. Furthermore, to accelerate the formation of myotubes more efficiently, the alginate/fibrin bioink with vascular endothelial cells was co-printed into a spatially patterned structure within a myoblast-laden structure. The myoblast-laden structure co-cultured with endothelial cells presented fully aligned myotube formation and significantly greater myogenic differentiation compared to the myoblast-laden structure without the endothelial cells owing to the more abundant secretion of angiogenic cytokines. Also, when adipose stem cell- and endothelial cell-laden fibrous structure was implanted in a mouse volumetric muscle loss model, accelerated volumetric muscle repair was observed compared to the defect model. Based on the results, this study demonstrates an alginate-based bioink and new bio-fabricating method to obtain microfibrous cell-laden alginate/fibrin structures with mechanically stable and topographical cues. The proposed method can provide a myoblast/endothelial cell-laden fibrous alginate structure to efficiently induce engineering of skeletal muscle tissue, which could be used in muscle-on-a-chip or recovering structures of volumetric muscle defects.

3D anisotropic conductive fibers electrically stimulated myogenesis

Recapitulation of in vivo environments that drive muscle cells to organize into a physiologically relevant 3D architecture remains a major challenge for muscle tissue engineering. To recreate electrophysiology of muscle tissues, electroactive biomaterials have been used to stimulate muscle cells with exogenous electrical fields. In particular, the use of electroactive biomaterials with an anisotropic micro-/nanostructure that closely mimic the native skeletal-muscle extracellular matrix (ECM) is desirable for skeletal muscle tissue engineering. Herein, we present a hierarchically organized, anisotropic, and conductive Polycaprolactone/gold (PCL/Au) scaffold for guiding myoblasts alignment and promoting the elongation and maturation of myotubes under electrical stimulation. Culturing with H9c2 myoblasts cells indicated that the nanotopographic cues was crucial for nuclei alignment, while the presence of microscale grooves effectively enhanced both the formation and elongation of myotubes. The anisotropic structure also leads to anisotropic conductivity. Under electrical stimulation, the elongation and maturation of myotubes were significantly enhanced along the anisotropic scaffold. Specifically, compared to the unstimulated group (0 V), the myotube area percentage increased by 1.4, 1.9 and 2.4 times in the 1 V, 2 V, 3 V groups, respectively. In addition, the myotube average length in the 1 V group increased by 1.3 times compared to that of the unstimulated group, and significantly increased by 1.8 and 2.0 times in the 2 V, 3 V groups, respectively. Impressively, the longest myotubes reached more than 4 mm in both 2 V and 3 V groups. Overall, our conductive, anisotropic 3D nano/microfibrous scaffolds with the application of electrical stimulation provides a desirable platform for skeletal muscle tissue engineering.

Biomimetic 3D aligned conductive tubular cryogel scaffolds with mechanical anisotropy for 3D cell alignment, differentiation and in vivo skeletal muscle regeneration

Developing 3D conductive aligned cryogels has great potential for skeletal muscle trauma treatment because they can mimic anisotropic structure, conductivity, and recoverable cyclic compression of the microenvironment of native skeletal muscle. In this work, a series of cryogels possessing 3D aligned morphology, conductivity, and excellent anisotropic mechanical compression property based on gelatin (GT) and polydopamine coated carbon nanotubes (PCNTs) were fabricated as skeletal muscle tissue scaffolds by using unidirectional freeze casting technology. The aligned microstructure of cryogels depended on gelatin concentration, and GT7.5 (with the gelatin content of 7.5% w/v) showed excellent aligned structure. Interestingly, the mechanical property of the aligned cryogels was similar to that of native skeletal muscle in terms of the dynamic contraction behavior and the anisotropic compression property due to the internal anisotropy structure. The aligned cryogel GT7.5 with good biocompatibility significantly promoted the alignment and elongation of C2C12 myoblasts. Moreover, the introduction of PCNTs enhanced the mechanical properties of cryogel GT7.5 and had a positive effect on myogenic differentiation of C2C12 cells. The aligned conductive GT7.5C2 cryogel significantly promoted new born muscle tissue generation compared to non-aligned group (GT7.5C2N) and non-conductive group (GT7.5) in a rat tibialis anterior muscle defect model. These data suggested that the 3D aligned conductive cryogel with conductivity and anisotropic compression property is a promising scaffold candidate for skeletal muscle tissue engineering.

Recycled algae-based carbon materials as electroconductive 3D printed skeletal muscle tissue engineering scaffolds

Skeletal muscle is an electrically and mechanically active tissue that contains highly oriented, densely packed myofibrils. The tissue has self-regeneration capacity upon injury, which is limited in the cases of volumetric muscle loss. Several regenerative therapies have been developed in order to enhance this capacity, as well as to structurally and mechanically support the defect site during regeneration. Among them, biomimetic approaches that recapitulate the native microenvironment of the tissue in terms of parallel-aligned structure and biophysical signals were shown to be effective. In this study, we have developed 3D printed aligned and electrically active scaffolds in which the electrical conductivity was provided by carbonaceous material (CM) derived from algae-based biomass. The synthesis of this conductive and functional CM consisted of eco-friendly synthesis procedure such as pre-carbonization and multi-walled carbon nanotube (MWCNT) catalysis. CM obtained from biomass via hydrothermal carbonization (CM-03) and its ash form (CM-03K) were doped within poly(ɛ-caprolactone) (PCL) matrix and 3D printed to form scaffolds with aligned fibers for structural biomimicry. Scaffolds were seeded with C2C12 mouse myoblasts and subjected to electrical stimulation during the in vitro culture. Enhanced myotube formation was observed in electroactive groups compared to their non-conductive counterparts and it was observed that myotube formation and myotube maturity were significantly increased for CM-03 group after electrical stimulation. The results have therefore showed that the CM obtained from macroalgae biomass is a promising novel source for the production of the electrically conductive scaffolds for skeletal muscle tissue engineering.

A mini review on 3-D bioprinting in skeletal muscle tissue engineering

This review focuses on advances in the field of bioprinting for skeletal tissue engineering, as well as a discussion of various bone marrow transplants. All methods (cellular, non-cellular and both) are aimed at finding complex living tissues capable of growing and maturing, using the same basic technology. To date, co-printing of cellular and non-cellular material has been identified as the most promising method for muscle applications because materials with good performance and good mechanical strength can be integrated within the same structure. Integration of orthopedic applications is an evolving field, and a detailed discussion of current challenges and ideas for the future is also presented in this review.

Transdifferentiation of Human Fibroblasts into Skeletal Muscle Cells: Optimization and Assembly into Engineered Tissue Constructs through Biological Ligands.

Simple SummaryEngineered human skeletal muscle tissue is a platform tool that can help scientists and physicians better understand human physiology, pharmacology, and disease modeling. Over the past few years this area of research has been actively being pursued by many labs worldwide. Significant challenges remain, including accessing an adequate cell source, and achieving proper physiological-like architecture of the engineered tissue. To address cell resourcing we aimed at further optimizing a process called transdifferentiation which involves the direct conversion of fibroblasts into skeletal muscle cells. The opportunity here is that fibroblasts are readily available and can be expanded sufficiently to meet the needs of a tissue engineering approach. Additionally, we aimed to demonstrate the applicability of transdifferentiation in assembling tissue engineered skeletal muscle. We implemented a screening process of protein ligands in an effort to refine transdifferentiation, and identified that most proteins resulted in a deficit in transdifferentiation efficiency, although one resulted in robust expansion of cultured cells. We were also successful in assembling engineered constructs consisting of transdifferentiated cells. Future directives involve demonstrating that the engineered tissues are capable of contractile and functional activity, and pursuit of optimizing factors such as electrical and chemical exposure, towards achieving physiological parameters observed in human muscle.The development of robust skeletal muscle models has been challenging due to the partial recapitulation of human physiology and architecture. Reliable and innovative 3D skeletal muscle models recently described offer an alternative that more accurately captures the in vivo environment but require an abundant cell source. Direct reprogramming or transdifferentiation has been considered as an alternative. Recent reports have provided evidence for significant improvements in the efficiency of derivation of human skeletal myotubes from human fibroblasts. Herein we aimed at improving the transdifferentiation process of human fibroblasts (tHFs), in addition to the differentiation of murine skeletal myoblasts (C2C12), and the differentiation of primary human skeletal myoblasts (HSkM). Differentiating or transdifferentiating cells were exposed to single or combinations of biological ligands, including Follistatin, GDF8, FGF2, GDF11, GDF15, hGH, TMSB4X, BMP4, BMP7, IL6, and TNF-α. These were selected for their critical roles in myogenesis and regeneration. C2C12 and tHFs displayed significant differentiation deficits when exposed to FGF2, BMP4, BMP7, and TNF-α, while proliferation was significantly enhanced by FGF2. When exposed to combinations of ligands, we observed consistent deficit differentiation when TNF-α was included. Finally, our direct reprogramming technique allowed for the assembly of elongated, cross-striated, and aligned tHFs within tissue-engineered 3D skeletal muscle constructs. In conclusion, we describe an efficient system to transdifferentiate human fibroblasts into myogenic cells and a platform for the generation of tissue-engineered constructs. Future directions will involve the evaluation of the functional characteristics of these engineered tissues.

Engineered neuromuscular actuators for medicine, meat, and machines

Movement is central to life. Neuromuscular tissues control voluntary movement in humans and many other living creatures, offering significant advantages in adaptability and robustness as compared to abiotic actuators. The impressive functional capabilities of neuromuscular tissues have inspired researchers to attempt de novo synthesis of the biological motor system via tissue engineering. This article highlights key recent advances in tissue engineering skeletal muscle and discusses promising strategies to control engineered muscle via biological neural networks and abiotic soft electronic interfaces. Challenges associated with cell sourcing, biomaterials design, and scalable precision manufacturing, along with emerging strategies to address those challenges, are presented. Finally, we highlight how engineered neuromuscular tissues have enabled studying, controlling, and deploying them as actuators in a range of real-world applications including drug discovery, regenerative medicine, cellular agriculture, and soft robotics.

Current Strategies for the Regeneration of Skeletal Muscle Tissue.

Traumatic injuries, tumor resections, and degenerative diseases can damage skeletal muscle and lead to functional impairment and severe disability. Skeletal muscle regeneration is a complex process that depends on various cell types, signaling molecules, architectural cues, and physicochemical properties to be successful. To promote muscle repair and regeneration, various strategies for skeletal muscle tissue engineering have been developed in the last decades. However, there is still a high demand for the development of new methods and materials that promote skeletal muscle repair and functional regeneration to bring approaches closer to therapies in the clinic that structurally and functionally repair muscle. The combination of stem cells, biomaterials, and biomolecules is used to induce skeletal muscle regeneration. In this review, we provide an overview of different cell types used to treat skeletal muscle injury, highlight current strategies in biomaterial-based approaches, the importance of topography for the successful creation of functional striated muscle fibers, and discuss novel methods for muscle regeneration and challenges for their future clinical implementation.

Decellularized grass as a sustainable scaffold for skeletal muscle tissue engineering.

Scaffold materials suitable for the scale-up and subsequent commercialization of tissue engineered products should ideally be cost effective and accessible. For the in vitro culture of certain adherent cells, synthetic fabrication techniques are often employed to produce micro- or nano-patterned substrates to influence cell attachment, morphology, and alignment via the mechanism of contact guidance. Here we present a natural scaffold, in the form of decellularized amenity grass, which retains its natural striated topography and supports the attachment, proliferation, alignment and differentiation of murine C2C12 myoblasts, without the need for additional functionalization. This presents an inexpensive, sustainable scaffold material and structure for tissue engineering applications capable of influencing cell alignment, a desired property for the culture of skeletal muscle and other anisotropic tissues.

Synthesis and characterization of C2C12-laden gelatin methacryloyl (GelMA) from marine and mammalian sources

Gelatin methacryloyl (GelMA) is widely used for tissue engineering applications as an extracellular matrix (ECM) mimicking scaffold due to its cost-effectiveness, ease of synthesis, and high biocompatibility. GelMA is widely synthesized from porcine skin gelatin, which labors under clinical, religious, and economical restrictions. In order to overcome these limitations, GelMA can be produced from fish skin gelatin, which is eco-friendly as well. Here, we present a comparative study of the physicochemical (structural, thermal, water uptake, swelling, rheological, and mechanical) and biological (cell viability, proliferation, and spreading) properties of porcine and fish skin GelMA with low and high methacrylation degrees, before and after crosslinking, to check whether fish skin can replace porcine skin as the source of GelMA. Porcine and fish skin GelMA presented similar structural, thermal, and water uptake properties prior to crosslinking. However, subsequent to crosslinking, fish skin GelMA hydrogels exhibited a higher mass swelling ratio and a lower elastic and compressive Young's moduli than porcine skin GelMA hydrogels of similar methacrylation level. Both types of GelMA hydrogels showed great biocompatibility toward encapsulated mouse myoblast cells (C2C12), however, improved cell spreading was observed in fish skin GelMA hydrogels, and cell proliferation was only induced in low methacrylated GelMA. These results suggest that fish skin GelMA is a promising substitute for porcine skin GelMA for biomedical applications and that low methacrylated fish skin GelMA can be used as a potential scaffold for skeletal muscle tissue engineering.

Modelling the growth of in-vitro meat on microstructured edible films

In-vitro meat is an emerging technology that involves the production of edible muscles using skeletal muscle tissue engineering avoiding in this way the slaughter of farmed animals. In the field of in-vitro meat production, it is important to tune appropriately the cell growth phase to obtain sufficient high biomass prior to entering to cell differentiation (nonproliferative stage). Moreover, the understanding of the kinetics associated with myoblast cell growth is necessary for scaling-up the production through the use of bioreactors. In this work, the growth of myoblasts paralleled aligned into an edible micropatterned film mimicking muscle fiber was modelled using the cubic Moser equation, where the oxygen diffusion into microchannels was calculated using a mass transfer approach. The availability of oxygen in contact with the muscle cells was lower in microchannels compared to a flat surface. These results demonstrate that microchannels offer to limit oxygen substrate to cultivate in-vitro meat. The results were consistent with observations of the cell growth onto two kinds of micropatterned edible films, which were fabricated using non-mammalian gelatin, alginate, agarose and glycerol or sorbitol as plasticizers, all of them already used as edible additives in the food industry.

Exploration of de novo‐ synthesized Zeolitic Imidazolate Framework‐8 (ZIF‐8) Nanoparticles as an Additive to Growth Media for Myoblasts

Skeletal muscle tissue engineering aims to repair a large area of damaged muscle caused by injuries or illness due to the difficulty of the cells naturally regenerating large tissue loss. Supplements help restore this loss via myoblast proliferation and maturation. Zinc excites the signal pathways for myogenesis, and as a supplement it should increase myoblast proliferation and maturation. The project goal was to synthesize nanoparticles that will stay suspended in cell media and allow the transfer of the zinc ion to increase muscle cell proliferation and maturation. Zeolitic imidazole framework-8 (ZIF-8) were created using a de novo synthesis, with the desired carboxaldehydes attached to the four imidazole rings surrounding a zinc ion core (Krishna et al. 2017). After synthesis, MP-AES, FT-IR, 13C-NMR, and 1H-NMR instruments were used to characterize the ZIF-8 nanoparticles. The synthesized product yields were 0.1098 g, 0.7400 g, and 0.1492 g, but the characterization data was inconsistent. 10 mg, 50 mg, and 100 mg of the synthesized products were then suspended in vials of 10 mL of phosphate buffered saline (PBS), sonicated for four, and six minutes with 20 seconds on and off, and the suspension time recorded. The 50 mg and 100 mg had the lowest suspension times (21 s to 162 s) while the 10 mg of product had higher suspension times (16 s to 384 s). In conclusion, the de novo ZIF-8 synthesis provided varying product yields; however, the suspendability times were too low. Ongoing research will focus on characterization, increasing the amount of zinc, and increasing the suspension time.

Exogenous FGF-2 prolongs endothelial connection in multilayered human skeletal muscle cell sheet

Angiogenesis is a pressing issue in tissue engineering associated with restoration of blood supply to ischemic tissues and promotion of rapid vascularization of tissue-engineered grafts. Fibroblast growth factor-2 (FGF-2) plays a vital role in processes such as angiogenesis and is an attractive candidate for tissue engineering. While skeletal muscle tissue engineering is established, the role of FGF-2 in endothelial function to promote angiogenesis after transplantation is unclear. Here, a culture system comprising a five-layered sheet of human skeletal muscle cells co-incubated on green fluorescent protein-expressing human umbilical vein endothelial cells (GFP-HUVECs) mimicking invivo angiogenesis was used to investigate the role of FGF-2 in vascularization of engineered tissues. The basal level of FGF-2 in cultured media of skeletal muscle cell sheets was undetectable. Therefore, cell sheets co-incubated with GFP-HUVECs were exogenously treated with 10ng/mL FGF-2, and endothelial network formation was evaluated. After prolonged culture, the endothelial network length and connectivity increased following treatment with FGF-2 as compared with control treatment. The numbers of medium and long endothelial networks significantly increased inside the sheet longer than 0.2 and 0.4cm, respectively, after FGF-2 treatment. Time-lapse microscopy monitoring dynamic endothelial behavior revealed that FGF-2-mediated maintenance of endothelial connection and retardation of endothelial network disconnection after 72h. The present study suggests the precise role of FGF-2 in maintaining endothelial connection and the extent of the endothelial network in skeletal muscle cell sheets. This understanding can be applied to design invitro pre-vascularized tissue and graft integration prospects.

Mechanobiology in Tendon, Ligament, and Skeletal Muscle Tissue Engineering.

Tendon, ligament, and skeletal muscle are highly organized tissues that largely rely on a hierarchical collagenous matrix to withstand high tensile loads experienced in activities of daily life. This critical biomechanical role predisposes these tissues to injury, and current treatments fail to recapitulate the biomechanical function of native tissue. This has prompted researchers to pursue engineering functional tissue replacements, or dysfunction/disease/development models, by emulating in vivo stimuli within in vitro tissue engineering platforms-specifically mechanical stimulation, as well as active contraction in skeletal muscle. Mechanical loading is critical for matrix production and organization in the development, maturation, and maintenance of native tendon, ligament, and skeletal muscle, as well as their interfaces. Tissue engineers seek to harness these mechanobiological benefits using bioreactors to apply both static and dynamic mechanical stimulation to tissue constructs, and induce active contraction in engineered skeletal muscle. The vast majority of engineering approaches in these tissues are scaffold-based, providing interim structure and support to engineered constructs, and sufficient integrity to withstand mechanical loading. Alternatively, some recent studies have employed developmentally inspired scaffold-free techniques, relying on cellular self-assembly and matrix production to form tissue constructs. Whether utilizing a scaffold or not, incorporation of mechanobiological stimuli has been shown to improve the composition, structure, and biomechanical function of engineered tendon, ligament, and skeletal muscle. Together, these findings highlight the importance of mechanobiology and suggest how it can be leveraged to engineer these tissues and their interfaces, and to create functional multitissue constructs.

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.

Fabrication of hybrid scaffolds obtained from combinations of PCL with gelatin or collagen via electrospinning for skeletal muscle tissue engineering.

The creation of skeletal muscle tissue in vitro is a major topic of interest today in the field of biomedical research, due to the lack of treatments for muscle loss due to traumatic accidents or disease. For this reason, the intrinsic properties of nanofibrillar structures to promote cell adhesion, proliferation, and cell alignment presents an attractive tool for regenerative medicine to recreate organized tissues such as muscle. Electrospinning is one of the processing techniques often used for the fabrication of these nanofibrous structures and the combination of synthetic and natural polymers is often required to achieve optimal mechanical and physiochemical properties. Here, polycaprolactone (PCL) is selected as a synthetic polymer used for the fabrication of scaffolds, and the effect of protein addition on the final scaffolds' properties is studied. Collagen and gelatin were the proteins selected and two different concentrations were analyzed (2 and 4 wt/vol%). Different PCL/protein systems were prepared, and a structural, mechanical and functional characterization was performed. The influence of fiber alignment on the properties of the final scaffolds was assessed through morphological, mechanical and biological evaluations. A bioreactor was used to promote cell proliferation and differentiation within the scaffolds. The results revealed that protein addition produced a decrease in the fiber size of the membranes, an increase in their hydrophilicity, and a softening of their mechanical properties. The biological study showed the ability of the selected systems to harbor cells, allow their growth and, potentially, develop musculoskeletal tissues.