BackgroundVolumetric 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.ResultsPrimary 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.ConclusionsIn 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-017-0131-2) contains supplementary material, which is available to authorized users.

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.

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Percent contribution of the thumb and the fingers to total grip strength differed between dominant and nondominant hands with a change in distribution when assessing maximum grip force. In right-handed subjects, thumb and ring finger have important roles during gripping.

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Background: Assessment of tissue perfusion after irradiation of random pattern flaps still remains a challenge. Methods: Twenty-five rats received harvesting of bilateral random pattern fasciocutaneous flaps. Group 1 served as nonirradiated control group. The right flaps of the groups 2–5 were irradiated with 20 Gy postoperatively (group 2), 3 × 12 Gy postoperatively (group 3), 20 Gy preoperatively (group 4) and 3 × 12 Gy preoperatively (group 5). Imaging with infrared thermography, indocyanine green angiography and near-infrared reflectance-based imaging were performed to detect necrotic areas of the flaps. Results: Analysis of the percentage of the necrotic area of the irradiated flaps showed a statistically significant increase from day 1 to 14 only in group 5 (p < 0.05). Indocyanine green angiography showed no differences (p > 0.05) of the percentage of the nonperfused area between all days in group 1 and 3, but a decrease in group 2 in both the left and the right flaps. Infrared thermography and near-infrared reflectance-based imaging did not show evaluable differences. Conclusion: Indocyanine green angiography is more precise in prediction of necrotic areas in random pattern skin flaps when compared to hyperspectral imaging, thermography or clinical impression. Preoperative fractional irradiation with a lower individual dose but a higher total dose has a more negative impact on flap perfusion compared to higher single stage irradiation.

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BackgroundSystemic thrombolysis with recombinant tissue plasminogen activator (rt-PA) is the standard of acute stroke care. Its potential to increase the risk of secondary intracerebral hemorrhage, especially if administered late, has been ascribed to its proteolytic activity that has detrimental effects on blood–brain barrier (BBB) integrity after stroke. FTY720 has been shown to protect endothelial barriers in several disease models such as endotoxin-induced pulmonary edema and therefore is a promising candidate to counteract the deleterious effects of rt-PA. Besides that, every putative neuroprotectant that will be eventually forwarded into clinical trials should be tested in conjunction with rt-PA.MethodsWe subjected C57Bl/6 mice to 3 h filament-induced tMCAO and postoperatively randomized them into four groups (n = 18/group) who received the following treatments directly prior to reperfusion: 1) vehicle-treatment, 2) FTY720 1 mg/kg i.p., 3) rt-PA 10 mg/kg i.v. or 4) FTY720 and rt-PA as a combination therapy. We measured functional neurological outcome, BBB disruption by quantification of EB extravasation and MMP-9 activity by gelatin zymography.ResultsWe observed a noticeable increase in mortality in the rt-PA/FTY720 cotreatment group (61%) as compared to the vehicle (33%), the FTY720 (39%) and the rt-PA group (44%). Overall, functional neurological outcome did not differ significantly between groups and FTY720 had no effect on rt-PA- and stroke-induced BBB disruption and MMP-9 activation.ConclusionsOur data show that FTY720 does not improve functional outcome and BBB integrity in large hemispheric infarctions, neither alone nor in conjunction with rt-PA. These findings stand in contrast to a recently published study that showed beneficial effects of FTY720 in combination with thrombolysis in a thrombotic model of MCAO leading to circumscript cortical infarctions. They might therefore represent a caveat that the coadministration of these two drugs might lead to excess mortality in the setting of a severe stroke.