Myogenic differentiation of primary myoblasts and mesenchymal stromal cells under serum-free conditions on PCL-collagen I-nanoscaffolds

Abstract

BackgroundThe creation of functional skeletal muscle via tissue engineering holds great promise without sacrificing healthy donor tissue. Different cell types have been investigated regarding their myogenic differentiation potential under the influence of various media supplemented with growth factors. Yet, most cell cultures include the use of animal sera, which raises safety concerns and might lead to variances in results. Electrospun nanoscaffolds represent suitable matrices for tissue engineering of skeletal muscle, combining both biocompatibility and stability.We therefore aimed to develop a serum-free myogenic differentiation medium for the co-culture of primary myoblasts (Mb) and mesenchymal stromal cells derived from the bone marrow (BMSC) and adipose tissue (ADSC) on electrospun poly-ε-caprolacton (PCL)-collagen I-nanofibers.ResultsRat Mb were co-cultured with rat BMSC (BMSC/Mb) or ADSC (ADSC/Mb) two-dimensionally (2D) as monolayers or three-dimensionally (3D) on aligned PCL-collagen I-nanofibers. Differentiation media contained either AIM V, AIM V and Ultroser® G, DMEM/Ham’s F12 and Ultroser® G, or donor horse serum (DHS) as a conventional differentiation medium. In 2D co-culture groups, highest upregulation of myogenic markers could be induced by serum-free medium containing DMEM/Ham’s F12 and Ultroser® G (group 3) after 7 days. Alpha actinin skeletal muscle 2 (ACTN2) was upregulated 3.3-fold for ADSC/Mb and 1.7-fold for BMSC/Mb after myogenic induction by group 3 serum-free medium when compared to stimulation with DHS. Myogenin (MYOG) was upregulated 5.2-fold in ADSC/Mb and 2.1-fold in BMSC/Mb. On PCL-collagen I-nanoscaffolds, ADSC showed a higher cell viability compared to BMSC in co-culture with Mb. Myosin heavy chain 2, ACTN2, and MYOG as late myogenic markers, showed higher gene expression after long term stimulation with DHS compared to serum-free stimulation, especially in BMSC/Mb co-cultures. Immunocytochemical staining with myosin heavy chain verified the presence of a contractile apparatus under both serum free and standard differentiation conditions.ConclusionsIn this study, we were able to myogenically differentiate mesenchymal stromal cells with myoblasts on PCL-collagen I-nanoscaffolds in a serum-free medium. Our results show that this setting can be used for skeletal muscle tissue engineering, applicable to future clinical applications since no xenogenous substances were used.

Proteins and Peptides as Important Modifiers of the Polymer Scaffolds for Tissue Engineering Applications—A Review

“…In recent years, biocompatible polymer scaffolds modified with proteins/peptides have been developed for regeneration of skin [192,202,203], nerves [204][205][206], muscles [207,208], tendons [209][210][211], cartilages [212][213][214], and bones [57,58,215]. To fabricate these biomaterials, many different techniques are applied.…”

“…Thus, porous scaffolds can be fabricated using freeze-drying, solvent casting/particulate leaching, or 3D printing, while fibrous biomaterials can be produced via electrospinning, self-assembly, phase separation, and solid free-form fabrication [4,9,17,19,200,216]. However, based on recent data, it seems that freeze-drying [57,[217][218][219] and electrospinning [58,192,207,215,220] are most frequently used. Freeze-drying, also referred to as lyophilization, is a simple method that allows obtaining 3D porous scaffolds.…”

Comparison and Optimization: Different Medium and a Novel Scheme for Rabbit Bone Marrow Mesenchymal Stem Cells Culture

“…The physiological characteristics of BMSCs are closely related to their culture environment. The presence or absence of serum [31,32], glucose content [24], related cytokines such as FGF [33], PH value [34] will all affect. Therefore, further investigations are required to develop a more satisfactory scheme for rBMSCs culture.…”

The Effect of Physical Cues of Biomaterial Scaffolds on Stem Cell Behavior

“…[ 49 ] However, it was found the topography of aligned electrospun nanofibers could induce myogenesis in standard growth medium (10% serum) for hMSCs, [ 50 ] or in serum‐free media for rat bone marrow‐derived MSCs (rBMSCs) or rat adipose‐derived stem cells (rADSCs) in coculture with rat myoblasts. [ 51 ] The aligned fibers resulted in cytoskeleton alignment, cellular, and nucleus elongation along the fibers, upregulation of myogenic gene expression, and improved myotube formation. Additional growth factors, such as transforming growth factor‐beta1 (TGF‐ β 1), [ 52 ] hepatocyte growth factor (HGF), or insulin‐like growth factor‐1 (IGF‐1) could have synergistic effects on myogenesis, but the topography of the scaffolds was believed to be more important.…”