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Understanding the Role of ECM Protein Composition and Geometric Micropatterning for Engineering Human Skeletal Muscle.

ABSTRACT: Skeletal muscle lost through trauma or disease has proven difficult to regenerate due to the challenge of differentiating human myoblasts into aligned, contractile tissue. To address this, we investigated microenvironmental cues that drive myoblast differentiation into aligned myotubes for potential applications in skeletal muscle repair, organ-on-chip disease models and actuators for soft robotics. We used a 2D in vitro system to systematically evaluate the role of extracellular matrix (ECM) protein composition and geometric patterning for controlling the formation of highly aligned myotubes. Specifically, we analyzed myotubes differentiated from murine C2C12 cells and human skeletal muscle derived cells (SkMDCs) on micropatterned lines of laminin compared to fibronectin, collagen type I, and collagen type IV. Results showed that laminin supported significantly greater myotube formation from both cells types, resulting in greater than twofold increase in myotube area on these surfaces compared to the other ECM proteins. Species specific differences revealed that human SkMDCs uniaxially aligned over a wide range of micropatterned line dimensions, while C2C12s required specific line widths and spacings to do the same. Future work will incorporate these results to engineer aligned human skeletal muscle tissue in 2D for in vitro applications in disease modeling, drug discovery and toxicity screening.

SUBMITTER: Duffy RM

PROVIDER: S-EPMC4880540 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Understanding the Role of ECM Protein Composition and Geometric Micropatterning for Engineering Human Skeletal Muscle.

Duffy Rebecca M RM Sun Yan Y Feinberg Adam W AW

Annals of biomedical engineering 20160316 6

Skeletal muscle lost through trauma or disease has proven difficult to regenerate due to the challenge of differentiating human myoblasts into aligned, contractile tissue. To address this, we investigated microenvironmental cues that drive myoblast differentiation into aligned myotubes for potential applications in skeletal muscle repair, organ-on-chip disease models and actuators for soft robotics. We used a 2D in vitro system to systematically evaluate the role of extracellular matrix (ECM) pr ...[more]

PMID: 26983843

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Project description:ObjectiveThe main objective of this study is to determine the myogenic effects of skeletal muscle extracellular matrix, vascular endothelial growth factor and human umbilical vein endothelial cells on adipose-derived stem cells to achieve a 3-dimensional engineered vascular-muscle structure.Materials and methodsThe present experimental research was designed based on two main groups, i.e. monoculture of adipose tissue-derived stem cells (ADSCs) and co-culture of ADSCs and human umbilical vein endothelial cells (HUVECs) in a ratio of 1:1. Skeletal muscle tissue was isolated, decellularized and its surface was electrospun using polycaprolactone/gelatin parallel nanofibers and then matrix topography was evaluated through H and E, trichrome staining and SEM. The expression of MyHC2 gene and tropomyosin protein were examined through real-time reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence, respectively. Finally, the morphology of mesenchymal and endothelial cells and their relationship with each other and with the engineered scaffold were examined by scanning electron microscopy (SEM).ResultsAccording to H and E and Masson's Trichrome staining, muscle tissue was completely decellularized. SEM showed parallel Polycaprolactone (PCL)/gelatin nanofibers with an average diameter of about 300 nm. The immunofluorescence proved that tropomyosin was positive in the ADSCs monoculture and the ADSCs/HUVECs coculture in horse serum (HS) and HS/VEGF groups. There was a significant difference in the expression of the MyHC2 gene between the ADSCs and ADSCs/HUVECs culture groups (P<0.05) and between the 2D and 3D models in HS/ VEGF differentiation groups (P<001). Moreover, a significant increase existed between the HS/VEGF group and other groups in terms of endothelial cells growth and proliferation as well as their relationship with differentiated myoblasts (P<0.05).ConclusionCo-culture of ADSCs/HUVECs on the engineered cell-free muscle scaffold and the dual effects of VEGF can lead to formation of a favorable engineered vascular-muscular tissue. These engineered structures can be used as an acceptable tool for tissue implantation in muscle injuries and regeneration, especially in challenging injuries such as volumetric muscle loss, which also require vascular repair.

Project description:Parameters of patients' body composition have been suggested as prognostic markers in several clinical conditions including cancer and liver transplantation, but only limited data on its value in critical illness exist to date. In this study, we aimed at evaluating a potential prognostic value of the skeletal muscle mass and skeletal muscle myosteatosis of critically ill patients at admission to the ICU.DesignExploratory observational cohort study.SettingAn urban, academic medical institution.PatientsOne-hundred fifty-five patients treated for critical illness on a medical ICU.InterventionsNone.Measurements and main resultsWe used routine CT scans to assess the patients' individual body composition. The skeletal muscle index as a surrogate for sarcopenia was defined as the total skeletal muscle area at the level of the third lumbar vertebra on axial CT scan, normalized for the patient's height. Myosteatosis was evaluated by assessing the mean skeletal muscle attenuation measured in Hounsfield unit at the same sectional plane. The skeletal muscle index and mean skeletal muscle attenuation at admission to the ICU were significantly higher in patients with long-term survival (180-day or 1-year mortality), while both parameters were comparable between short-term survivors and nonsurvivors (ICU mortality or 30-d mortality). Patients with a skeletal muscle index or mean skeletal muscle attenuation below our established ideal cutoff values (74.95 mm2/cm and 29 Hounsfield unit) showed a significantly reduced overall survival. These findings were confirmed in univariate and multivariate Cox regression analyses. Furthermore, myosteatosis significantly correlated with the time of mechanical ventilation, the duration of hospital stay, and the presence of sepsis.ConclusionsOur data suggest that sarcopenia and myosteatosis represent important prognostic factors in critically ill patients that can be easily obtained from routine CT scans. Both parameters at admission to the ICU yield important information on the patients' long-term outcome and might be used for early clinical decision-making in these patients.

Dataset Information

Understanding the Role of ECM Protein Composition and Geometric Micropatterning for Engineering Human Skeletal Muscle.

Publications

Understanding the Role of ECM Protein Composition and Geometric Micropatterning for Engineering Human Skeletal Muscle.

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