The influence of electrospun aligned poly(ɛ-caprolactone)/collagen nanofiber meshes on the formation of self-aligned skeletal muscle myotubes
Introduction
Skeletal muscle defects due to trauma or tumor ablation usually require reconstructive procedures in order to restore normal tissue function. Currently, muscle pedicle flap from adjacent regions is the primary method practiced. However, this option is challenged by the host muscle tissue availability and donor site morbidity such as functional loss and volume deficiency [1], [2]. Recent advances in cell therapy using myoblasts have provided an alternate therapeutic opportunity to regenerate muscle tissue for functional augmentation [3], [4]. Injection of cultured myoblasts has shown some efficacy. However, this approach may not be suitable for treating large muscle tissue defects. Creation of implantable functional muscle tissue that could restore muscle tissue defects may be a possible solution [5], [6], [7].
An essential step in engineering functional skeletal muscle tissue is to mimic the structure of native tissue which is comprised of highly oriented myofibers formed from numerous fused mononucleated muscle cells. It is well known that structure and organization of muscle fibers dictate tissue function. Thus, muscle cell alignment that permits organized myotube formation is a crucial step in the musculoskeletal myogenesis [8]. The ability to efficiently organize muscle cells to form aligned myotubes in vitro would greatly benefit efforts in skeletal muscle tissue engineering.
To achieve an appropriate cellular organization for muscle tissue, the interaction between cells and surfaces of biomaterials is considered important, and understanding of cellular behaviors such as cell adhesion, proliferation, and migration is necessary. It is widely accepted that cell adhesion and proliferation of different cell types on substrates depend on the surface characteristics such as wettability, chemistry, electric charge, and topography of surfaces [9], [10], [11], [12], [13]. Moreover, chemical and topographic substrate surface patterning have been used as a method to control cellular organization [14]. Consequently, surface characteristics of biomaterials play an important role in guiding cellular organization.
Skeletal muscle tissue is composed of bundles of highly oriented and densely packed muscle fibers, each with multinucleated cells derived from myoblasts. The fibers are densely packed together in extracellular matrix (ECM) to constitute an organized muscle tissue that generates longitudinal contraction [15], [16]. To engineer functional muscle tissue for reconstruction, scaffolds that permit cellular organization that more closely mimics native individual fiber formation with unidirectional orientation are needed. Electrospinning has been widely used as a fabrication method to generate nanofibers with a diameter range of several micrometers to 100 nm or less for various tissue applications [17]. The nano-scaled fiber structures generated by this method are able to support cell adhesion and guide cellular behavior [18], [19]. In addition, this technology offers the ability to control scaffold composition, structure, and mechanical properties [20], [21]. We previously have demonstrated that three-dimensional electrospun nanofibers scaffolds with high porosity and interconnectivity provide a favorable environment for cells using a variety of synthetic and naturally derived biomaterials [20].
Nanofiber scaffolds generated by electrospinning should have appropriate material characteristics for skeletal muscle. They should be biocompatible to permit cell adhesion and growth, degradable over time as muscle cells mature into tissue, and elastic to accommodate contractile function. Poly(ɛ-caprolactone) (PCL) is a degradable polyester which is known to possess these characteristics. We previously have demonstrated that PCL can be fabricated into nanofibers and that it can be combined with type I collagen to achieve scaffolds that are more biocompatible and improve cellular adhesion and growth characteristics [22]. In this study we examined the feasibility of using PCL/collagen based nanofibers using electrospinning as a scaffold system for implantable engineered muscle. We investigated whether orientation of electrospun PCL/collagen nanofibers influences morphology, adhesion, proliferation, differentiation, and organization of human skeletal muscle cells (hSkMCs), and promote myotube formation.
Section snippets
Materials
Collagen type I derived from calfskin was purchased from Elastin Products Company (Owensville, MO, USA). Poly(ɛ-caprolactone) (PCL, Inherent viscosity: 1.77 dL/g in CHCl3 at 30 °C) was obtained from Lactel Absorbable Polymers (Pelham, AL, USA). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFP) and all chemicals were purchased from Sigma Co. (St Louis, MO, USA) and used as received unless stated otherwise.
Electrospinning
Electrospun nanofiber meshes were fabricated using a blend of PCL and collagen with a ratio of 1:1 in
Fabrication and characterization of the electrospun PCL/collagen nanofiber meshes
PCL/collagen blend solution was electrospun onto a stainless steel plate at different rotation speeds to form various fiber orientations in the nanofibers. SEM images of the resulting fibers showed nano-scaled fiber diameters and controlled fiber orientations (Fig. 2a–d). The electrospun meshes of the PCL/collagen blend were produced from solutions having a total polymer concentration ranging from 3 to 10% (wt/vol) in HFP. The electrospun meshes showed a linear relationship between solution
Discussion
To more closely approximate native skeletal muscle architecture, this study was designed to fabricate nanofiber meshes that are aligned in a unidirectional manner to facilitate myotube formation. We evaluated cell adhesion, proliferation, and differentiation on the electrospun nanofiber meshes with different fiber orientations using primary hSkMCs. In this study we show that oriented nanofiber meshes, composed of PCL and type I collagen, can be fabricated using electrospinning technology. Human
Conclusions
PCL/collagen nanofiber meshes fabricated by electrospinning show a unidirectional fiber orientation that can guide hSKMCs alignment and enhance myotube formation. The electrospun meshes are biocompatible, biodegradable, easily fabricated, and are able to support cell adhesion, proliferation, and differentiation. This study suggests that aligned PCL/collagen nanofibers facilitate skeletal muscle cell organization, which may result in improved muscle function and maturation.
Acknowledgements
We would like to thank Dr. Jennifer Olson for editorial assistance, Dr. John J. Smith III for assistance of muscle biopsy, and Cathy M. Mathis for technical assistance with immunohistochemistry.
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