PORTEFEUILLE

Peripheral nerve injuries are frequent, causing substantial work leave, chronic disabling and healthcare expenses. Microsurgical repair is the current treatment of choice. However, the clinical outcome is often disappointing, because of axonal misrouting and the long time required for regeneration, resulting in poor re-innervation of the target organs (motor and sensory). In this project, we attempt to promote peripheral nerve regeneration by using biodegradable nerve conduits (NC) from which glial cell-derived neurotrophic factor (GDNF) is released at controlled kinetics. The aim is to increase significantly the speed of axonal regeneration and the functional quality of target re-innervation of a severed peroneal nerve in the rat.
This medical bioengineering project is expected to make a substantial contribution to the understanding of the role of defined GDNF delivery kinetics from biodegradable NC on axonal growth and nerve regeneration. This gained knowledge should bear consequences in the therapeutic use of GDNF and other nerve growth factors.

This transdisciplinary project is located at the interface between natural sciences, engineering and medical sciences. It relies on competencies in the areas of biomaterials, drug formulation and delivery, and reconstructive (hand, oto-rhino-laryngology) surgery. The project is well integrated in the main research areas of all co-investigators, i.e., formulation and controlled delivery of growth factors at ETH Zürich, studies on peripheral nerve regeneration and microsurgical nerve repair at CHUV and the Center for Hand Surgery and Therapy. In addition to the transdisciplinary cooperation between the scientists directly involved, the project engages the commitment of pharmaceutical companies.
The successful outcome of the project should influence the common approach in nerve repair that uses mostly autologous material or medical devices with purely passive properties. The proposed combination of a nerve conduit (medical device) with a separately customizable delivery system for GDNF (medicinal product) is highly innovative, should create added therapeutic value and be cost-effective from a societal point of view.
The project is self-contained and should provide new impulses for novel therapeutic approaches relying on both medical devices and highly potent drugs. The focus put on the release kinetics of GDNF and the impact of these variations on axonal regeneration and maturation is quite an original approach. The combination of medical devices and drugs has not been much exploited so far, because of limited interactions between the pharmaceutical and medical device researchers and industry.
The transfer potential from pre-clinical research to clinical research is particularly high, as the study is embedded in close proximity to clinical environment and performed by clinicians. Thus, successful growth stimulatory NC prototypes may be rapidly used in selected patients for demonstrating the clinical feasibility of the approach.

Biodegradable growth-stimulatory nerve conduits (NC) were produced from: (i) alginate-chitosan complexes, (ii) collagen, and (iii) silk, by spinning mandrel technology. The NC pos-sessed adequate mechanical properties in terms of tensile and compression strength, stress at break, and surgical handling for sewing. All NC-types exhibited good biocompatibility. Non-etheless, as the alginate-chitosan complexes caused substantial infiltration of macrophages upon implantation in rats, this material was abandoned for further development.
The growth stimulatory signals encompassed the two neurotrophic factors (NTFs) GDNF and NGF, and, in the case of silk NC, aligned silk nanofibers that were inserted into the NC. Delivery of the neurotrophic factors from the NC was prolonged over 4 weeks, period during which the released proteins remained bioactive. In vitro, NGF promoted mostly axonal branch-ing, whereas GDNF promoted primarily the axonal elongation, as demonstrated with DRG ex-plants from chicken embryos. GDNF and NGF acted synergistically on the enhancement of both the rate and length of axonal outgrowth. The important synergistic effect of GDNF and NGF was also confirmed in vivo with collagen NC implanted into a sciatic nerve gap in the rat. Here, the combination of GDNF and NGF enhanced significantly the length of regenerating axons, Schwann cells migration, total number of axons, axon diameter, axon density, number of myelinated axons, myelin area, and whole nerve area, as compared to all other treatment conditions (GDNF alone; control NTF-free NC; NGF alone was not tested). Another important finding was the dependency of the axonal growth in vivo on the extent of immediate GDNF release. High initial burst release of NTFs from the NC hampered significantly nerve regen-eration as compared to low burst release, although the extent of burst release did not affect the diameter of the axons.
A further enhancement of the functionality of future NC may consist in the integration of autologous Schwann cells, which are modified with transgene encoding for NTFs. In view of this approach, initial experiments were made for transduction of Schwann cells from rats by recombinant viral vectors expressing NTFs. In future work, the benefit of the Schwann cells with modified gene expression of NTFs over added drug substance-based growth factor deliv-ery will be assessed in the context of NC. The main benefit of Schwann cells over pure growth factors may consist of the larger spectrum of natural growth enhancing stimuli released by the cells and the potential of proliferation and migration of the cells along with the regenerating axons.

Übersax L, Mattotti M, Papaloïzos M, Merkle HP, Gander B, Meinel L, Silk fibroin matrices for the controlled release of nerve growth factor (NGF). Biomaterials 28, 4449-4460 (2007).
Piquilloud G, Christen T, Pfister L, Gander B, Papaloïzos MY, Variations in glial cell-line de-rived neurotrophic factor release from biodegradable nerve conduits modify the rate of functional motor recovery after art primary nerve repairs. Eur. J Neurosci. 26, 1109-1117 (2007).
Madduri S, Papaloïzos M, Gander B, Synergistic effect of GDNF and NGF on axonal branch-ing and elongation in vitro. Neurosci. Res 64, (2009). (in press; online preview: doi:10.1016/j.neures.2009.06.003).
Madduri S, di Summa PG, Papaloïzos M, Kalbermatten D, Gander B, Synergistic effect of GDNF and NGF on nerve regeneration in vivo. (in preparation).
Maddurui S, Wandrey Meinel A, Papaloïzos M, Gander B. Trophically and tropically function-alized silk fibroin nerve conduits for guided peripheral nerve regeneration. (in preparation).
Madduri S, Feldman K, Tervoort T, Papaloïzos M, Gander B. Collagen nerve conduits releas-ing the neurotrophic factors GDNF and NGF. (in preparation)

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