Mesenchymal stem cells and myoblast differentiation under HGF and IGF-1 stimulation for 3D skeletal muscle tissue engineering.

BACKGROUND: Volumetric muscle loss caused by trauma or after tumour surgery exceeds the natural regeneration capacity of skeletal muscle. Hence, the future goal of tissue engineering (TE) is the replacement and repair of lost muscle tissue by newly generating skeletal muscle combining different cell sources, such as myoblasts and mesenchymal stem cells (MSCs), within a three-dimensional matrix. Latest research showed that seeding skeletal muscle cells on aligned constructs enhance the formation of myotubes as well as cell alignment and may provide a further step towards the clinical application of engineered skeletal muscle. In this study the myogenic differentiation potential of MSCs upon co-cultivation with myoblasts and under stimulation with hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) was evaluated. We further analysed the behaviour of MSC-myoblast co-cultures in different 3D matrices. RESULTS: Primary rat myoblasts and rat MSCs were mono- and co-cultivated for 2, 7 or 14 days. The effect of different concentrations of HGF and IGF-1 alone, as well as in combination, on myogenic differentiation was analysed using microscopy, multicolour flow cytometry and real-time PCR. Furthermore, the influence of different three-dimensional culture models, such as fibrin, fibrin-collagen-I gels and parallel aligned electrospun poly-ε-caprolacton collagen-I nanofibers, on myogenic differentiation was analysed. MSCs could be successfully differentiated into the myogenic lineage both in mono- and in co-cultures independent of HGF and IGF-1 stimulation by expressing desmin, myocyte enhancer factor 2, myosin heavy chain 2 and alpha-sarcomeric actinin. An increased expression of different myogenic key markers could be observed under HGF and IGF-1 stimulation. Even though, stimulation with HGF/IGF-1 does not seem essential for sufficient myogenic differentiation. Three-dimensional cultivation in fibrin-collagen-I gels induced higher levels of myogenic differentiation compared with two-dimensional experiments. Cultivation on poly-ε-caprolacton-collagen-I nanofibers induced parallel alignment of cells and positive expression of desmin. CONCLUSIONS: In this study, we were able to myogenically differentiate MSC upon mono- and co-cultivation with myoblasts. The addition of HGF/IGF-1 might not be essential for achieving successful myogenic differentiation. Furthermore, with the development of a biocompatible nanofiber scaffold we established the basis for further experiments aiming at the generation of functional muscle tissue.

A Novel Simple Approach to Material Parameters from Commonly Accessible Rheometer Data.

When characterizing the viscoelastic properties of polymers, shear rheological measurements are commonly the method of choice. These properties are known to affect extrusion and nozzle-based processes such as fiber melt spinning, cast film extrusion and 3D-printing. However, an adequate characterization of shear thinning polymers can be challenging and still insufficient to not only describe but predict process relevant influences. Furthermore, the evaluation of rheological model systems in literature is mostly based on stress-relaxation experiments, which are rarely available for various polymeric materials. Therefore, a simple approach is presented, that can be used to evaluate and benchmark a wide range of rheological model systems based on commonly accessible frequency sweep data. The approach is validated by analyzing alginate PH176 solutions of various concentrations, a thermoplastic poly-urethane (TPU) Elastollan 1180A melt, the liquid silicon rubber Elastosil 7670 and a polycaprolactone (PCL) fiber-alginate composite system. The used rheological model systems, consisting of simple springs and dashpots, are suitable for the description of complex, viscoelastic material properties that can be observed for polymer solutions and gel-like systems. After revealing a suitable model system for describing those material properties, the determination and evaluation of relevant model parameters can take place. We present a detailed guideline for the systematic parameter revelation using alginate solutions of different concentrations as example. Furthermore, a starting point for future correlations of strut spreading in 3D-bioprinting and model parameters is revealed. This work establishes the basis for a better understanding and potential predictability of key parameters for various fabrication techniques.

Novel PGS/PCL electrospun fiber mats with patterned topographical features for cardiac patch applications.

Nano- and micro-scale topographical features play a critical role in the induction and maintenance of various cellular properties and functions, including morphology, adhesion, gene regulation, and cell-to-cell communication. In addition, recent studies have indicated that the structure and function of heart tissue are also sensitive to mechanical cues at the nano- and micro-scale. Although fabrication methods exist for generating topographical features on polymeric scaffolds for cell culture, current techniques, especially those with nano-scale resolution, are typically complex, prohibitively expensive and not accessible to most biology laboratories. Here, we present a simple and tunable fabrication method for the production of patterned electrospun fibers that simulate the complex anisotropic and multi-scale architecture of cardiac tissue, to promote cardiac cell alignment. This method is based on the combination of electrospinning and soft lithography techniques, in which electrospun fibers, based on a blend of poly(glycerol sebacate) and poly(caprolactone), were collected on a patterned Teflon-coated silicon wafer with imprinted topographical features. Different surface topographies were investigated, such as squares and grooves, with constant or different interspatial distances. In vitro cell culture studies successfully demonstrated the alignment of both C2C12 myoblasts and neonatal rat cardiomyocytes on fabricated electrospun patterned surfaces. C2C12 cells were cultured over a period of 72h to study the effect of topographical cues on cell morphology. Cells attached within the first 8h after seeding and after 24h most of the cells started to align responding to the topographical cues. Similarly, cardiomyocytes responded to the topographical features by aligning themselves and by expressing Connexin 43 along cellular junctions. Summarizing, we have developed a new method with the potential to significantly promote cardiac tissue engineering by fabricating electrospun fibers with defined topographical features to guide and instruct donor and/or host cells.

AC electrophoretic deposition of organic-inorganic composite coatings.

Alternating current electrophoretic deposition (AC-EPD) of polyacrylic acid (PAA)-titanium oxide (TiO(2)) nanoparticle composites on stainless steel electrodes was investigated in basic aqueous solution. AC square wave with duty cycle of 80% was applied at a frequency of 1 kHz. FTIR-ATR spectra showed that both AC and direct current (DC) EPD successfully deposited PAA-TiO(2) composites. The deposition rate using AC-EPD was lower than that obtained in direct current DC-EPD. However, the microstructure and surface morphology of the deposited composite coatings were different depending on the type of electric field applied. AC-EPD applied for not more than 5 min led to smooth films without bubble formation, while DC-EPD for 1 min or more showed deposits with microstructural defects possibly as result of water electrolysis. AC-EPD was thus for the first time demonstrated to be a suitable technique to deposit organic-inorganic composite coatings from aqueous suspensions, showing that applying a square wave and frequency of 1 kHz leads to uniform PAA-TiO(2) composite coatings on conductive materials.

Novel poly(hydroxyalkanoates)-based composites containing Bioglass® and calcium sulfate for bone tissue engineering.

Three different poly(hydroxyalkanoates) (PHAs), copolymers of poly(3-hydroxybutyrate) (P3HB), have been used to make composites using two different fillers, bioactive glass (type 45S5 Bioglass®) and calcium sulfate dihydrate. The PHAs used were poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [PHBHV] and two copolymers of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [PHBHHx]. The aim of the study was the fabrication and characterization of the new composites and the assessment of the influence of the particular filler combination on the physical properties and bioactivity of the films. The thermal behaviour was studied using differential scanning calorimetry while mechanical properties were evaluated using dynamic mechanic thermal analysis and tensile strength test. The mechanical and thermal properties were affected by particles addition. The distribution of the particles in the polymer matrix, observed by scanning electron microscopy, was directly related to the mechanical properties. The surface characteristics were investigated by contact angle measurements and Raman spectroscopy. The extent of formation of hydroxyapatite (HA) upon immersion in simulated body fluid (SBF) depended on the polymer used, the amount of fillers employed and the time of immersion in SBF. Bioactivity was enhanced in the composites with a rise of hydrophilicity. The HA formation was controllable with time in the case of PHBHHx composites.