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

BACKGROUND: The 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. RESULTS: Rat 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. CONCLUSIONS: In 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.

Investigation of the batch-to-batch inconsistencies of Collagen in PCL-Collagen nanofibers.

The application of tissue engineered constructs is an approach in regenerative therapies to support regeneration of damaged muscular tissue. Therefore the development of highly aligned electrospun scaffolds based on polycaprolactone (PCL) and collagen enables a versatile development towards tailor made applications. However, the application of natural polymers like collagen brings the risk of batch-to-batch inconsistencies, which influence the reproducibility of the electrospinning process. Aligned PCL-Collagen nanofibers were fabricated via electrospinning using benign solvents. The spinnability of different collagen batches and polymer concentrations in diluted acetic acid as solvent was investigated. Furthermore spinning parameters and fiber morphology were investigated in order to determine the most stable spinning conditions and analyze the batch-to-batch variations. Finally the effect of the solution temperature and the time of pure collagen in solution were investigated, to complete the analysis of the influences on the spinning behavior.

Incorporation of Cu-Containing Bioactive Glass Nanoparticles in Gelatin-Coated Scaffolds Enhances Bioactivity and Osteogenic Activity.

Bioactive glass scaffolds (BGS) of 45S5 composition exhibit desired bioactivity, osteogenesis, and angiogenesis potential, being promising biomaterials for bone repair/regeneration. Natural polymer-based coatings, e.g., gelatin coating, are effective to enhance the mechanical properties of BGS. However, the presence of a coating may reduce the bioactivity and osteogenesis activity of the scaffolds. To address the issue of reduced osteogenic properties induced by polymer coatings, in this study, we incorporated Cu-containing bioactive glass nanoparticles (Cu-BGN: 95SiO2-2.5CaO-2.5CuO, in mol %), as bioactive fillers, into the gelatin coating. The bioactivity (apatite-forming ability) of the gelatin coated BGS was improved after the incorporation of Cu-BGN in the coating. Hydroxyapatite could form on the Cu-BGN/gelatin nanocomposite coated BGS within 1 day of immersion in simulated body fluid. The osteogenic activity as indicated by the ALP activity of MC3T3-E1 cells on the coated BGS was also significantly enhanced after the incorporation of Cu-BGN. In addition, the incorporation of Cu-BGN in the coating did not affect the highly porous and interconnected pore structure of BGS while the mechanical improvement induced by the gelatin coating remained after the addition of Cu-BGN. The attachment of MC3T3-E1 cells on the scaffolds was not influenced by the presence of Cu-BGN in the gelatin coating, while the cell proliferation was enhanced. In conclusion, the incorporation of bioactive nanoparticles into polymer coating is presented as a solution to the reduced bioactivity and osteogenic activity of polymer coated 45S5 BGS. The Cu-BGN/gelatin nanocomposite coated BGS exhibiting high bioactivity, appropriate mechanical properties, and osteogenic potential are candidate biomaterials for bone tissue engineering/regeneration.

Novel approach towards aligned PCL-Collagen nanofibrous constructs from a benign solvent system.

Under several conditions such as severe trauma skeletal muscle lack the ability to reorganize and the loss of muscle function is inevitable. The application of tissue engineered constructs is a promising approach in regenerative therapies for damaged muscular tissue. Therefore, the development of highly aligned scaffolds based on polycaprolactone (PCL) has been studied extensively. Nanofiber scaffolds containing collagen have mostly been fabricated via electrospinning using highly corrosive 1,1,1,3,3,3 hexafluoro-2-propanol (HFIP) so far. In this study, aligned Polycaprolactone-Collagen (PCL-Coll) biocomposite nanofibers were fabricated via electrospinning using environmentally benign diluted acetic acid (AcOH) as solvent. Furthermore, ultrasonic treatment was introduced to enhance the intrinsically weak solubility of PCL in AcOH. AcOH was diluted to an ideal concentration for electrospinning of 90%. The final solutions were spun at various conditions and collected with different collector setups in order to determine ideal processing conditions for the fabrication of highly aligned nanofibers.

Vascular Tissue Engineering: Effects of Integrating Collagen into a PCL Based Nanofiber Material.

The engineering of vascular grafts is a growing field in regenerative medicine. Although numerous attempts have been made, the current vascular grafts made of polyurethane (PU), Dacron®, or Teflon® still display unsatisfying results. Electrospinning of biopolymers and native proteins has been in the focus of research to imitate the extracellular matrix (ECM) of vessels to produce a small caliber, off-the-shelf tissue engineered vascular graft (TEVG) as a substitute for poorly performing PU, Dacron, or Teflon prostheses. Blended poly-ε-caprolactone (PCL)/collagen grafts have shown promising results regarding biomechanical and cell supporting features. In order to find a suitable PCL/collagen blend, we fabricated plane electrospun PCL scaffolds using various collagen type I concentrations ranging from 5% to 75%. We analyzed biocompatibility and morphological aspects in vitro. Our results show beneficial features of collagen I integration regarding cell viability and functionality, but also adverse effects like the loss of a confluent monolayer at high concentrations of collagen. Furthermore, electrospun PCL scaffolds containing 25% collagen I seem to be ideal for engineering vascular grafts.

Development and characterization of novel electrically conductive PANI-PGS composites for cardiac tissue engineering applications.

Cardiovascular diseases, especially myocardial infarction, are the leading cause of morbidity and mortality in the world, also resulting in huge economic burdens on national economies. A cardiac patch strategy aims at regenerating an infarcted heart by providing healthy functional cells to the injured region via a carrier substrate, and providing mechanical support, thereby preventing deleterious ventricular remodeling. In the present work, polyaniline (PANI) was doped with camphorsulfonic acid and blended with poly(glycerol-sebacate) at ratios of 10, 20 and 30vol.% PANI content to produce electrically conductive composite cardiac patches via the solvent casting method. The composites were characterized in terms of their electrical, mechanical and physicochemical properties. The in vitro biodegradability of the composites was also evaluated. Electrical conductivity increased from 0Scm(-1) for pure PGS to 0.018Scm(-1) for 30vol.% PANI-PGS samples. Moreover, the conductivities were preserved for at least 100h post fabrication. Tensile tests revealed an improvement in the elastic modulus, tensile strength and elasticity with increasing PANI content. The degradation products caused a local drop in pH, which was higher in all composite samples compared with pure PGS, hinting at a buffering effect due to the presence of PANI. Finally, the cytocompatibility of the composites was confirmed when C2C12 cells attached and proliferated on samples with varying PANI content. Furthermore, leaching of acid dopants from the developed composites did not have any deleterious effect on the viability of C2C12 cells. Taken together, these results confirm the potential of PANI-PGS composites for use as substrates to modulate cellular behavior via electrical stimulation, and as biocompatible scaffolds for cardiac tissue engineering applications.