TE (tissue engineering) of skeletal muscle is a promising method to reconstruct loss of muscle tissue. This study evaluates MSCs (mesenchymal stem cells) as new cell source for this application. As a new approach to differentiate the MSCs towards the myogenic lineage, co-cultivation with primary myoblasts has been developed and the myogenic potential of GFP (green fluorescent protein)-transduced rat MSC co-cultured with primary rat myoblasts was assessed by ICC (immunocytochemistry). Myogenic potential of MSC was analysed by ICC, FACS and qPCR (quantitative PCR). MSC-myoblast fusion phenomena leading to hybrid myotubes were evaluated using a novel method to evaluate myotube fusion ratios based on phase contrast and fluorescence microscopy. Furthermore, MSC constitutively expressed the myogenic markers MEF2 (myogenic enhancer factor 2) and α-sarcomeric actin, and MEF2 expression was up-regulated upon co-cultivation with primary myoblasts and the addition of myogenic medium supplements. Significantly higher numbers of MSC nuclei were involved in myotube formations when bFGF (basic fibroblast growth factor) and dexamethasone were added to co-cultures. In summary, we have determined optimal co-culture conditions for MSC myogenic differentiation up to myotube formations as a promising step towards applicability of MSC as a cell source for skeletal muscle TE as well as other muscle cell-based therapies.

BackgroundTissue engineering of vascularised skeletal muscle is a promising method for the treatment of soft tissue defects in reconstructive surgery. In this study we explored the characteristics of novel collagen and fibrin matrices for skeletal muscle tissue engineering. We analyzed the characteristics of newly developed hybrid collagen-I-fibrin-gels and collagen nanofibers as well as collagen sponges and OPLA®-scaffolds. Collagen-fibrin gels were also tested with genipin as stabilizing substitute for aprotinin.ResultsWhereas rapid lysis and contraction of pure collagen I- or fibrin-matrices have been great problems in the past, the latter could be overcome by combining both materials. Significant proliferation of cultivated myoblasts was detected in collagen-I-fibrin matrices and collagen nanofibers. Seeding cells on parallel orientated nanofibers resulted in strongly aligned myoblasts. In contrast, common collagen sponges and OPLA®-scaffolds showed less cell proliferation and in collagen sponges an increased apoptosis rate was evident. The application of genipin caused deleterious effects on primary myoblasts.ConclusionCollagen I-fibrin mixtures as well as collagen nanofibers yield good proliferation rates and myogenic differentiation of primary rat myoblasts in vitro In addition, parallel orientated nanofibers enable the generation of aligned cell layers and therefore represent the most promising step towards successful engineering of skeletal muscle tissue.

Muscle tissue engineering (TE) has not yet been clinically applied because of several problems. However, the field of skeletal muscle TE has been developing tremendously and new approaches and techniques have emerged. This review will highlight recent developments in the field of nanotechnology, especially electrospun nanofibre matrices, as well as potential cell sources for muscle TE. Important developments in cardiac muscle TE and clinical studies on Duchenne muscular dystrophy (DMD) will be included to show their implications on skeletal muscle TE.

Treatment of complex bone defects in which vascular supply is insufficient is still a challenge. To overcome the limitations from autologous grafts, a sheep model has been established recently, which is characterized by the development of an independent axial vascularization of a bioartificial construct, permitting microsurgical transplantation. To engineer independently axially vascularized bone tissue in the sheep arteriovenous (AV)-loop model, mesenchymal stem cells (MSCs), without and in combination with recombinant human bone morphogenetic protein-2 (rhBMP-2), were harvested and directly autotransplanted in combination with β-tricalcium phosphate-hydroxyapatite (β-TCP-HA) granules into sheep in this study. After explantation after 12 weeks, histological and immunohistochemical evaluation revealed newly formed bone in both groups. An increased amount of bone area was obtained using directly autotransplanted MSCs with rhBMP-2 stimulation. Osteoblastic and osteoclastic cells were detected adjacent to the newly formed bone, revealing an active bone remodelling process. Directly autotransplanted MSCs can be found close to the β-TCP-HA granules and are contributing to bone formation. Over time, magnetic resonance imaging (MRI) and micro-computed tomography (μCT) imaging confirmed the dense vascularization arising from the AV-loop. This study shows de novo engineering of independently axially vascularized transplantable bone tissue in clinically significant amounts, using directly autotransplanted MSCs and rhBMP-2 stimulation in about 12 weeks in the sheep AV-loop model. This strategy of engineering vascularized transplantable bone tissue could be possibly transferred to the clinic in the future in order to augment current reconstructive strategies.

Nanofiber scaffolds have proven their various advantages for tissue engineering and have been analyzed extensively. However, to date the three-dimensional pattern of vascularization inside nanofibrous scaffolds is unknown. This study introduces a novel method to visualize and quantify vascularization of electrospun nanofibrous PCL/collagen scaffolds in 3D in vivo. Randomly spun PCL/collagen blend and parallel aligned PCL/collagen blend/PEO scaffolds were analyzed for numbers and patterns of sprouting vessels inside the constructs using microCT scans at different time points. The image data derived from the microCT scans was converted into three-dimensional vessel trees. The aligned scaffold showed a significantly smaller number of sprouting vessels but vascularization in the center of the constructs occurred considerably earlier than in the nonwoven scaffold. Thus, for the first time the actual pattern of vascularization in nanofibrous scaffolds can be visualized three-dimensionally. These results demonstrate that the 3D pattern of vessel trees could be an essential parameter to evaluate nanofiber scaffolds for their suitability for tissue engineering as well as in vivo applications in general.