Electronmicroscopical evaluation of short-term nerve regeneration through a thin-walled biodegradable poly(DLLA-ε-CL) nerve guide filled with modified denatured muscle tissue
Introduction
Transplantation of autologous nerve grafts is now the most widely used technique in microsurgery when direct anastomosis of the nerve stumps is impossible. The results, however, are disappointing. The procedure requires sacrifice of a donor nerve, mostly a sensory nerve. In addition, there is also the risk of neuroma formation at the donor site.
Biodegradable nerve guides provide a successful alternative [1], [2]. The idea behind the use of a biodegradable nerve guide is, that it directs the outgrowing nerve fibers towards the distal nerve stump, whilst preventing neuroma formation and ingrowth of fibrous tissue into the nerve gap. After serving these functions, the nerve guide should gradually degrade, without inducing scar tissue formation.
The use of a biodegradable nerve guide composed of an amorphous copolymer of dl-lactide and ε-caprolactone [p(DLLA-ε-CL)] has proven to be effective and promising [3], [4], [5]. Nerve regeneration across a 1 cm nerve gap, using a biodegradable nerve guide, was faster and qualitatively better, when compared with nerve regeneration through an autologous nerve graft [6]. Moreover, this nerve guide degrades quickly and completely within 1 year [5], and reconstruction with this nerve guide showed good functional nerve recovery [7].
To bridge a nerve gap of several centimeters (a more realistic point of view in the clinical situation), the addition of nerve growth stimulation factors will be necessary. Several researchers have already studied the influence of growth factors [10], extracellular matrix molecules [11], [12], [13], and freeze–thawed muscle tissue [14] on peripheral nerve regeneration. Most factors positively influence peripheral nerve regeneration.
The use of denatured muscle tissue inside a nerve guide is very promising, since the longitudinally oriented basal lamina of the muscle tissue will direct the outgrowing nerve fibers towards the distal nerve stump by functioning as a scaffold for the outgrowing nerves [15]. Recently, we evaluated different preparation techniques of denatured muscle tissue [16], aiming at an open structure of the extracellular matrix (ECM) and an intact basement membrane. In that study, we showed that the basal lamina contained both collagen type IV and laminin, which are known to enhance the outgrowth and regeneration of peripheral nerve fibers [17]. Besides the positive influence on peripheral nerve regeneration, the presence of denatured muscle tissue inside the nerve guide might prevent collapse of the nerve guides. In another study, we evaluated functional nerve recovery in the rat after reconstruction of a 15-mm nerve gap using a thin-walled nerve guide filled with MDMT [18]. We concluded that the use of MDMT increased the speed of recovery after reconstruction of a nerve gap with a p(DLLA-ε-CL) biodegradable nerve guide. However, the results obtained in that study (recovery of functionality) could not be directly related to speed and quality of nerve regeneration. To evaluate this relationship, transmission electron microscopy (TEM) and morphometric analysis of the axon regeneration process should therefore be carried out.
Den Dunnen et al. showed that after implantation of subcutaneous p(DLLA-ε-CL) bars swelling of the biomaterial during degradation occurred [8], which can hamper the nerve regeneration [3]. In this study, a nerve guide with an internal diameter of 1.4 mm and a wall thickness of approximately 0.17 mm was used. In a previous study, this so-called thin-walled nerve guide was used for the reconstruction of a 10-mm gap in the sciatic nerve of the rat and showed no macroscopical swelling [9]. However, most nerve guides collapsed.
The aim of this study was to evaluate nerve regeneration through a thin-walled biodegradable poly(dl-lactide-ε-caprolactone) nerve guide filled with MDMT. To do so, we evaluated regenerating nerves using transmission electron microscopy after implantation periods ranging from 3 to 12 weeks. To obtain more objective data for statistics we performed morphometric analysis of the regeneration process.
Section snippets
Nerve guides and modified denatured muscle tissue
The biodegradable nerve guide in this study was composed of a copolymer of 50% dl-lactide and 50% ε-caprolactone. The lactide component contained 85% l-lactide and 15% d-lactide. The nerve guide had an internal diameter of 1.4 mm and a wall thickness of 0.17 mm. The characteristics of the biomaterial and preparation techniques have been described in detail by den Dunnen et al. [4]. Before implantation, the nerve guides were filled with modified denatured muscle tissue with the basal lamina
Results
Nerve regeneration occurred in all rats. The orientation of the regenerating nerve fibers was good, neuroma formation and collapsing of the nerve guides was not observed.
Discussion
Nerve regeneration is significantly compromized when large nerve gaps have to be bridged. The maximum gap length that can be bridged with empty nerve guides is limited, depending on the kind of biomaterial that is used for the tube and the porosity of the biomaterial [20], [21]. Before bridging a nerve gap of several centimeters, a nerve guide filled with MDMT is tested. When only a thin-walled nerve guide is used to bridge a nerve gap, the nerve guide will collapse [9]. This phenomenon was
Acknowledgments
This research was made possible by the MW-NWO (Dutch Organization for Scientific Research), Den Haag, The Netherlands. The assistance of H.L. Bartels for the microsurgical techniques is greatly appreciated. The authors gratefully acknowledge P. van der Sijde, D. Huizinga, and B. Hellinga for photography, and Miss M. Heekelaar for her assitance in morphometric analysis. This work was sponsored by “The Cock Foundation”, The Netherlands.
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