New aspects on efficient anticoagulation and antiplatelet strategies in sheep.

BACKGROUND: After addressing fundamental questions in preclinical models in vitro or in small animals in vivo, the translation into large animal models has become a prerequisite before transferring new findings to human medicine. Especially in cardiovascular, orthopaedic and reconstructive surgery, the sheep is an important in vivo model for testing innovative therapies or medical devices prior to clinical application. For a wide variety of sheep model based research projects, an optimal anticoagulation and antiplatelet therapy is mandatory. However, no standardised scheme for this model has been developed so far. Thus the efficacy of antiplatelet (acetylsalicylic acid, clopidogrel, ticagrelor) and anticoagulant (sodium enoxaparin, dabigatran etexilate) strategies was evaluated through aggregometry, anti-factor Xa activity and plasma thrombin inhibitor levels in sheep of different ages. RESULTS: Responses to antiplatelet and anticoagulant drugs in different concentrations were studied in the sheep. First, a baseline for the measurement of platelet aggregation was assessed in 20 sheep. The effectiveness of 225 mg clopidogrel twice daily (bid) in 2/5 sheep and 150 mg bid in 3/5 lambs could be demonstrated, while clopidogrel and its metabolite carboxylic acid were detected in every plasma sample. High dose ticagrelor (375 mg bid) resulted in sufficient inhibition of platelet aggregation in 1/5 sheep, while acetylsalicylic acid did not show any antiplatelet effect. Therapeutic anti-factor Xa levels were achieved with age-dependent dosages of sodium enoxaparin (sheep 3 mg/kg bid, lambs 5 mg/kg bid). Administration of dabigatran etexilate resulted in plasma concentrations similar to human ranges in 2/5 sheep, despite receiving quadruple dosages (600 mg bid). CONCLUSION: High dosages of clopidogrel inhibited platelet aggregation merely in a low number of sheep despite sufficient absorption. Ticagrelor and acetylsalicylic acid cannot be recommended for platelet inhibition in sheep. Efficient anticoagulation can be ensured using sodium enoxaparin rather than dabigatran etexilate in age-dependent dosages. The findings of this study significantly contribute to the improvement of a safe and reliable prophylaxis for thromboembolic events in sheep. Applying these results in future translational experimental studies may help to avoid early dropouts due to thromboembolic events and associated unnecessary high animal numbers.

Myogenic differentiation of mesenchymal stem cells co-cultured with primary myoblasts.

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.

Factors influencing successful outcome in the arteriovenous loop model: a retrospective study of 612 loop operations.

The arteriovenous (AV) loop is a complex model and requires advanced microsurgical skills. After several years of studies including characterization of the physiological and molecular phenomena behind the process of neovascularization, we would like to evaluate our experience from a microsurgical point of view. The AV loop operation was performed in 612 male Lewis rats. Species, surgeon, previous microsurgical skills, solid or soft consistency of matrix, weeks from implantation to explantation, experience, and practice of surgeon as well length of operation and outcome (patent versus thrombosed) upon evaluation were analyzed for each loop operation. Previous microsurgical skills, matrix, and explantation interval were not significant predictors. There was a significant correlation of experience, practice, and individual performance of microsurgical technicians with the rate of thrombosis and duration of the procedure. Experimental microsurgical operations on animals are best performed by especially trained technicians, rather than microsurgeons working parallel to their clinical activities. The AV loop is an excellent model in the arena of translational technologies, but due to its complexity is unsuitable for screening purposes. Experience and practice are both significant predictors of successful outcome. Possible applications of the model for research purposes are discussed.

Engineering skeletal muscle tissue--new perspectives in vitro and in vivo.

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.

Collagen matrices from sponge to nano: new perspectives for tissue engineering of skeletal muscle.

BACKGROUND: Tissue 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. RESULTS: Whereas 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. CONCLUSION: Collagen 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.

Myogenic differentiation of mesenchymal stem cells in a newly developed neurotised AV-loop model.

Generation of axially vascularized muscle tissue constitutes a promising new approach to restoration of damaged muscle tissue. Mesenchymal stemcells (MSC), with their ability to be expanded to large cell numbers without losing their differentiation capacity into the myogenic lineage, could offer a promising cell source to generate neomuscle tissue. In vitro experiments showed that cocultures of primary myoblasts and MSC undergo myogenic differentiation by stimulation with bFGF and dexamethasone. A newly developed AV-Loop model with neurotization was established in this study. It encompasses axial vascularization and the additional implantation of a motor nerve serving as myogenic stimulator. Myoblasts and MSCs were coimplantated in a prevascularized isolation chamber. Cells were differentiated by addition of bFGF and dexamethasone plus implantation of a motor nerve. After 8 weeks, we could observe areas of myogenic differentiation with α -sarcomeric actin and MHC expression in the constructs. Quantitative PCR analysis showed an expression of myogenic markers in all specimens. Thus, neurotization and addition of bFGF and dexamethasone allow myogenic differentiation of MSC in an axially vascularized in vivo model for the first time. These findings are a new step towards clinical applicability of skeletal muscle tissue engineering and display its potential for regenerative medicine.

Engineering axially vascularized bone in the sheep arteriovenous-loop model.

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 ß-tricalcium phosphate-hydroxyapatite (ß-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 ß-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.

Three-dimensional vascularization of electrospun PCL/collagen-blend nanofibrous scaffolds in vivo.

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.