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Silk fibroin scaffolds with muscle-like elasticity support in vitro differentiation of human skeletal muscle cells.

ABSTRACT: Human adult skeletal muscle has a limited ability to regenerate after injury and therapeutic options for volumetric muscle loss are few. Technologies to enhance regeneration of tissues generally rely upon bioscaffolds to mimic aspects of the tissue extracellular matrix (ECM). In the present study, silk fibroins from four Lepidoptera (silkworm) species engineered into three-dimensional scaffolds were examined for their ability to support the differentiation of primary human skeletal muscle myoblasts. Human skeletal muscle myoblasts (HSMMs) adhered, spread and deposited extensive ECM on all the scaffolds, but immunofluorescence and quantitative polymerase chain reaction analysis of gene expression revealed that myotube formation occurred differently on the various scaffolds. Bombyx mori fibroin scaffolds supported formation of long, well-aligned myotubes, whereas on Antheraea mylitta fibroin scaffolds the myotubes were thicker and shorter. Myotubes were oriented in two perpendicular layers on Antheraea assamensis scaffolds, and scaffolds of Philosamia/Samia ricini (S. ricini) fibroin poorly supported myotube formation. These differences were not caused by fibroin composition per se, as HSMMs adhered to, proliferated on and formed striated myotubes on all four fibroins presented as two-dimensional fibroin films. The Young's modulus of A. mylitta and B. mori scaffolds mimicked that of normal skeletal muscle, but A. assamensis and S. ricini scaffolds were more flexible. The present study demonstrates that although myoblasts deposit matrix onto fibroin scaffolds and create a permissive environment for cell proliferation, a scaffold elasticity resembling that of normal muscle is required for optimal myotube length, alignment, and maturation. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd. StartCopTextStartCopText© 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

SUBMITTER: Chaturvedi V

PROVIDER: S-EPMC5724504 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Silk fibroin scaffolds with muscle-like elasticity support in vitro differentiation of human skeletal muscle cells.

Chaturvedi Vishal V Naskar Deboki D Kinnear Beverley F BF Grenik Elizabeth E Dye Danielle E DE Grounds Miranda D MD Kundu Subhas C SC Coombe Deirdre R DR

Journal of tissue engineering and regenerative medicine 20161122 11

Human adult skeletal muscle has a limited ability to regenerate after injury and therapeutic options for volumetric muscle loss are few. Technologies to enhance regeneration of tissues generally rely upon bioscaffolds to mimic aspects of the tissue extracellular matrix (ECM). In the present study, silk fibroins from four Lepidoptera (silkworm) species engineered into three-dimensional scaffolds were examined for their ability to support the differentiation of primary human skeletal muscle myobla ...[more]

PMID: 27878977

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Project description:Mechanical loading plays a major role in bone remodeling and fracture healing. Mimicking the concept of mechanical loading of bone has been widely studied in bone tissue engineering by perfusion cultures. Nevertheless, there is still debate regarding the in-vitro mechanical stimulation regime. This study aims at investigating the effect of two different flow rates (vlow = 0.001m/s and vhigh = 0.061m/s) on the growth of mineralized tissue produced by human mesenchymal stromal cells cultured on 3-D silk fibroin scaffolds. The flow rates applied were chosen to mimic the mechanical environment during early fracture healing or during bone remodeling, respectively. Scaffolds cultured under static conditions served as a control. Time-lapsed micro-computed tomography showed that mineralized extracellular matrix formation was completely inhibited at vlow compared to vhigh and the static group. Biochemical assays and histology confirmed these results and showed enhanced osteogenic differentiation at vhigh whereas the amount of DNA was increased at vlow. The biological response at vlow might correspond to the early stage of fracture healing, where cell proliferation and matrix production is prominent. Visual mapping of shear stresses, simulated by computational fluid dynamics, to 3-D micro-computed tomography data revealed that shear stresses up to 0.39mPa induced a higher DNA amount and shear stresses between 0.55mPa and 24mPa induced osteogenic differentiation. This study demonstrates the feasibility to drive cell behavior of human mesenchymal stromal cells by the flow velocity applied in agreement with mechanical loading mimicking early fracture healing (vlow) or bone remodeling (vhigh). These results can be used in the future to tightly control the behavior of human mesenchymal stromal cells towards proliferation or differentiation. Additionally, the combination of experiment and simulation presented is a strong tool to link biological responses to mechanical stimulation and can be applied to various in-vitro cultures to improve the understanding of the cause-effect relationship of mechanical loading.

Project description:Three-dimensional porous scaffolds prepared from regenerated silk fibroin using either an all-aqueous process or a process involving an organic solvent, hexafluoroisopropanol (HFIP), have shown promise in cell culture and tissue engineering applications. However, their biocompatibility and in vivo degradation have not been fully established. The present study was conducted to systematically investigate how processing method (aqueous vs. organic solvent) and processing variables (silk fibroin concentration and pore size) affect the short-term (up to 2 months) and long-term (up to 1 year) in vivo behavior of the protein scaffolds in both nude and Lewis rats. The samples were analyzed by histology for scaffold morphological changes and tissue ingrowth, and by real-time RT-PCR and immunohistochemistry for immune responses. Throughout the period of implantation, all scaffolds were well tolerated by the host animals and immune responses to the implants were mild. Most scaffolds prepared from the all-aqueous process degraded to completion between 2 and 6 months, while those prepared from organic solvent (hexafluoroisopropanol (HFIP)) process persisted beyond 1 year. Due to widespread cellular invasion throughout the scaffold, the degradation of aqueous-derived scaffolds appears to be more homogeneous than that of HFIP-derived scaffolds. In general and especially for the HFIP-derived scaffolds, a higher original silk fibroin concentration (e.g. 17%) and smaller pore size (e.g. 100-200microm) resulted in lower levels of tissue ingrowth and slower degradation. These results demonstrate that the in vivo behavior of the three-dimensional silk fibroin scaffolds is related to the morphological and structural features that resulted from different scaffold preparation processes. The insights gained in this study can serve as a guide for processing scenarios to match desired morphological and structural features and degradation time with tissue-specific applications.

Project description:The dissolution of Bombyx mori silk fibroin (SF) films in formic acid (FA) for the preparation of electrospinning dopes is widely exploited to produce electrospun SF scaffolds. The SILKBridge® nerve conduit is an example of medical device having in its wall structure an electrospun component produced from an FA spinning dope. Though highly volatile, residual FA remains trapped into the bulk of the SF nanofibers. The purpose of this work is to investigate the type and strength of the interaction between FA and SF in electrospun mats, to quantify its amount and to evaluate its possible toxicological impact on human health. The presence of residual FA in SF mats was detected by FTIR and Raman spectroscopy (new carbonyl peak at about 1,725 cm-1) and by solid state NMR, which revealed a new carbonyl signal at about 164.3 ppm, attributed to FA by isotopic 13C substitution. Changes occurred also in the spectral ranges of hydroxylated amino acids (Ser and Thr), demonstrating that FA interacted with SF by forming formyl esters. The total amount of FA was determined by HS-GC/MS analysis and accounted for 247 ± 20 μmol/g. The greatest part was present as formyl ester, a small part (about 3%) as free FA. Approximately 17% of the 1,500 μmol/g of hydroxy amino acids (Ser and Thr) theoretically available were involved in the formation of formyl esters. Treatment with alkali (Na2CO3) succeeded to remove the greatest part of FA, but not all. Alkali-treated electrospun SF mats underwent morphological, physical, and mechanical changes. The average diameter of the fibers increased from about 440 nm to about 480 nm, the mat shrunk, became stiffer (the modulus increased from about 5.5 MPa to about 7 MPa), and lost elasticity (the strain decreased from about 1 mm/mm to about 0.8 mm/mm). Biocompatibility studies with human adult dermal fibroblasts did not show significant difference in cell proliferation (313 ± 18 and 309 ± 23 cells/mm2 for untreated and alkali-treated SF mat, respectively) and metabolic activity. An in-depth evaluation of the possible toxicological impact of residual FA was made using the SILKBridge® nerve conduit as case study, following the provisions of the ISO 10993-1 standard. The Potential Patient Daily Intake, calculated from the total amount of FA determined by HS-GC/MS, was 2.4 mg/day and the Tolerable Exposure level was set to 35.4 mg/day. This allowed to obtain a value of the Margin of Safety of 15, indicating that the amount of FA left on SF mats after electrospinning does not raise concerns for human health.

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Silk fibroin scaffolds with muscle-like elasticity support in vitro differentiation of human skeletal muscle cells.

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Silk fibroin scaffolds with muscle-like elasticity support in vitro differentiation of human skeletal muscle cells.

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