Biomaterials 25 (2004) 2585–2594 Multilayered peptide incorporated collagen tubules for peripheral nerve repair M. Rafiuddin Ahmed, U. Venkateshwarlu, R. Jayakumar* Bio-organic and Neurochemistry Laboratory, Central Leather Research Institute, Adyar, Chennai 600 020, India Received 2 April 2003; accepted 4 September 2003 Abstract Successful nerve regeneration process was achieved with improved mechanical strength by crosslinking tubular nerve guides made up of collagen. The multilayered collagen sheets were prepared from laminar evaporation of collagen solution. Scanning electron micrograph of the collagen tubes crosslinked with glutaraldehyde (GTA), microwave irradiation showed porous, fibrillar structures of collagen filaments in these matrices. The mechanical property of the crosslinked collagen tubes was carried out by tensile strength measurements. Fourier transform infrared spectra of the collagen films show that the native triple helicity was unaltered during multilayered preparation. It was observed that the structural integrity is unaltered during the multilayer preparation. Microscopic analysis indicates that the tubule surface acts as a surface of adherence and proliferation for the sprouting axons from the cut proximal nerve stumps. Solute diffusion studies on these tubes indicate that they are highly porous to wide range of molecular sizes during regeneration. Among the two types of crosslinking, the microwave irradiated collagen conduits results in ample myelinated axons compared with GTA group, where we observed more unmyelinated axons. r 2003 Elsevier Ltd. All rights reserved. Keywords: Collagen; Peripheral nerve regeneration; Crosslinking; Contact guidance; Irradiation; Tubule permeability 1. Introduction The functional recovery of a peripheral nerve requires not only regeneration of nervous tissue across the gap but also an elongation of axons through the distal segment and arrival at their normal termini [1]. In short nerve injuries this is facilitated by using the end-to-end fascicular suture method, and for extensive nerve injuries a nerve bridge technique is preferred [2–4]. Various synthetic, polymeric and biological materials have been used to serve the function of a bridging source [5–10]. Collagen, which is used in peripheral nerve repair [11] as a bridging source has the uniqueness of less antigenicity and easy resorption in the body [12]. Stability and amenability to crosslink with various crosslinking agents, collagen is more advantageous than other types of biomolecules. Crosslinking agents like formaldehyde, glutaraldehyde (GTA), epoxy com- pounds [13], UV rays and g-irradiation methods were used in the fabrication of collagen based biomaterials [14]. Recently, Itoh et al. [15] studied about effective crosslinking of collagen with UV irradiation for peripheral nerve repair. Among the crosslinking agents, GTA is frequently used because of its high solubility in aqueous solution [16]. The parameters like surface properties, porosity and biodegradation will affect cell substrate interaction or communication with the ex- ternal environment of the conduits. Hence, optimiza- tions of these parameters are to be carried out to develop efficient nerve conduits with distinct character- istics in nerve tissue engineering [17]. Further cross- linking also prevents physical swelling of proteinous matrix thereby preventing the destruction of the tubular structure before the regeneration is complete. Other than stability, the nerve conduits are to be functionalized for better anchorage of regenerating fibres inside the nerve guides. This could be achieved by incorporating glycosaminoglycan complexes [18], laminin, fibronectin [19], dialysed plasma [20] as neurotrophic agents into silicone and collagen tubing. However, a non-absorbable silicone tubule results in scar formation, sheath stenosis [21]. Further removal of ARTICLE IN PRESS *Corresponding author. Tel.: +91-44-24911386; fax: +91-44- 24911589. E-mail address: karkuvi77@yahoo.co.uk (R. Jayakumar). 0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2003.09.075