Development of a longitudinal nerve con-duit matrix from autologous plasma for guided nerve regeneration

Status:

completed 12/2015

Aims:

Treatment of peripheral nerve defects using autograft is not always possible and alternatives are offered in the field of regenerative cell therapy and biomaterials. The use of an autologous plasma matrix as a filler for nerve conduits is promising as only autologous material is used and additional allogeneic or xenogeneic additives are not necessary.

In this in vitro study a longitudinally aligned fibrin matrix on the basis of a plasma clot was designed in order to guide regenerating nerve cells directionally through conduits.

Activities/Methods:

see results

Results:

Platelet-free plasma was used for the production of the plasma clot matrix, which was obtained by centrifugation of citrated blood from volunteers. The clot polymerization was carried for one hour at room temperature using different experimental conditions: a static control (without further manipulation), application of an electric field and under unidirectional movement (flow conditions). Originally undirected fibrin fibers were readily aligned during the clotting-process under the experimental conditions, but not in the control as shown by a self-developed nerve conduit model. Fiber alignment was analyzed by means of scanning electron microscopy (SEM) and confocal laser scanning microscopy (LSM). Output images were processed using fast Fourier transformation (FFT). Full width at half maximum (FWHM) and peak area (Xmin to Xmax) as well as the alignment index of the FFT-graph plots were calculated to determine the relative degree of fiber alignment.

Control clots formed a randomly organized, relatively homogenous fibrin network. In contrast, under flow conditions as well as in the electric field, clots showed longitudinal-orientated fibrin fibers. The orientation of the fibers was parallel to the flow direction or to the electrical-field lines. Added neural stem cells (H9) or mesenchymal stem cells (MSC) remained viable and exhibited a longitudinal orientation along the aligned fibrin fibers.

Furthermore, a migration model has been developed on the basis of an aligned or non-aligned plasma clot to simulate a nerve-conduit with integrated fibrin fibers and a chemotaxis assay was established using PMN, MSC and H9 cells. The formation of the chemokine gradient in the plasma clot matrix was demonstrated with the fluorescent dye FITC-dextran. Subsequently, the sensitivity of the cells to different potentially chemotactic factors was analyzed and quantified. The optimized fibrin matrices were finally tested using commercial clinically approved chitosan conduits. Results showed that the combination of hollow nerve conduits with an autologous directed plasma clot matrix is a potential new method for promoting peripheral nerve regeneration.

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