Manufacture of porous polymer nerve conduits by a novel low-pressure injection molding process

Abstract

A method to fabricate porous, biodegradable conduits using a combined injection molding, thermally induced phase transition technique was developed which produced conduits with dimensionally toleranced, longitudinally aligned channels. The geometry of the channels was designed to approximate the architecture of peripheral nerves and to support the monolayer adherence of physiologically relevant numbers of Schwann cells. The channel configuration could be varied from a single 1.35 mm diameter channel up to 100 0.08 mm diameter channels. A conduit with 100 channels has approximately 12.5 times the lumenal surface area of a single channel conduit and supports the adherence of five times the number of Schwann cells in the native peripheral nerve. In this study, poly(dl-lactide-co-glycolide) (dl-PLGA) was dissolved in acetic acid and injected into a cold mold which induced solid–liquid phase separation and, ultimately, solidification of the polymer solution. The acetic acid was removed by sublimation and the resulting foam had a macrostructure of high anisotropy. Semi-permeable skins formed on the outer and lumen diameters of the conduit as a consequence of rapid quenching. Macropores were organized into bundles of channels, up to 20 μm wide, in the dl-PLGA matrix and represented remnants of acetic acid that crystallized during solidification.

Introduction

The capacity of axons to regenerate within injured peripheral nerves has long been recognized. Despite microsurgical technique advances and the attainment of cellular and molecular insight into the process of regeneration, functional recovery of the injured peripheral nerve is rarely complete; epineurial suture of autologous nerve grafts remains the most widely used method for repair of peripheral nerve defects [1]. Nerve autografts are not ideal repair conduits, as the function of donor nerves is sacrificed without achievement of full functional recovery of the repaired nerve.

Research has focused on the creation of optimal scaffolds to serve as nerve guidance conduits between the proximal and distal nerve stumps ends. These guidance conduits must direct axons sprouting from the proximal regenerating nerve end, provide a conduit for diffusion of neurotropic and neurotrophic factors secreted by the damaged nerve stump, minimize the infiltration of fibrous tissue, retain adequate mechanical strength and flexibility to support the regenerating nerve fibers, and be biocompatible and biodegradable, so as to be integrated into the surrounding tissue after complete regeneration [2].

Many studies have focused on devising nerve guidance conduits from natural, biological materials. Improved regeneration with respect to controls has been demonstrated from autogenous materials such as skeletal muscle basal lamina grafts, with complex basal lamina architecture, and vein grafts [3], [4], [5], [6], [7]. Regeneration has been modest through these conduits, and harvesting these materials has resulted in donor site morbidity.

Several biodegradable synthetic materials, in the form of simple hollow conduits, have been shown to support nerve regeneration. Polyesters, such as polylactic acid (PLA), polyglycolic acid (PGA), and PLGA [8], [9], [10] have been used extensively because of their availability, ease of processing, approval by the FDA, and low inflammatory response. Other biodegradable polyesters have also demonstrated promise for nerve regeneration applications, such as poly(lactide-ε-caprolactone) [11], [12], [13], biodegradable polyurethanes [14], poly(organo)phosphazenes [15], and, most recently, trimethylene carbonate-co-ε-caprolactone [16], [17], [18].

In addition to biodegradability and biocompatibility issues, the influence of a variety of physical parameters of the conduits has been examined. Nerve regeneration has been enhanced with respect to controls by proper choice of hollow-tube characteristics, such as conduit diameter and length [19], lumenal surface microgeometry [20], [21], and wall porosity and permeability [22], [23], [24], [25]. Incorporation of an oriented intraluminal framework into the conduit lumen, which qualitatively models biological autogenous materials, has been a significant advancement; these conduits have demonstrated superior regeneration because the intraluminal matrix supports cell attachment/migration and guides regenerating axons. A variety of aligned matrix strategies have been investigated, such as: autologous vein conduits or synthetic conduits with inserted acellular muscle [26], [27], macroporous synthetic foams with oriented or interconnected pores [28], synthetic or collagen-based filaments within or without conduits [29], [30], [31], [32], [33], [34], collagen sponges within synthetic conduits [34], magnetically aligned collagen gels [35], and silicon with micromachined holes of various diameters and spacings [36], [37], [38].

Permeable hollow wall conduits have been produced using a variety of processing technologies. Porous mesh or foam sheets have been rolled and sealed, either by welding [39], suturing [40], [41], [42], glueing [43], [44], joining with solvent [45] or bonding with polymer solution [46]. Porous biodegradable hollow tube conduits have also been manufactured by dip-coating a mandrel into a polymer suspension containing water-soluble porogen particles, such as sugar or sodium chloride, which are leached out post-processing [47], [48], [49]. Conduits have been formed by extrusion of a polymer/salt composite [50] or by immersion precipitation, where a polymer non-solvent effects phase separation of a polymer solution, followed by subsequent gelation to immobilize the microporous structure [51].

In addition to conduit modifications, manipulating the internal environment of nerve conduits has improved nerve regeneration by the supplementation of extracellular matrix molecules, growth factors, and Schwann cells [52], [53]. Schwann cells serve as a living source of neurotropic and neurotrophic factors for the regenerating nerve, excrete extracellular matrix, and act as a substrate for elongating axons. Enhanced regeneration has been observed when Schwann cells were seeded into nerve conduits [54], [55], [56], [57], [58], [59], [60], [61], [62], largely as suspended Schwann cells. However, axonal growth is guided by Schwann cell columns in the Büngner bands during regeneration. To mimic this configuration, several studies have examined the impact of presenting Schwann cells as adherent monolayers, rather than as suspended cells. In most cases, the Schwann cells were attached only to the lumenal surface of the hollow tube nerve conduit, so that few Schwann cells were introduced [56], [59], [62], and the effect on regeneration was minimal. However, Shen et al. [32] recently described the design of a nerve conduit in which Schwann cells were presented adherent to matrigel coated Vicryl and polydioxanone filaments inserted into hollow tube conduits; Schwann cell chains were observed in structures similar to Büngner bands.

The objective of this study was to manufacture and characterize porous biodegradable polymer nerve conduits for guided tissue regeneration. These conduits contained longitudinally aligned channels and mimicked the geometry of autografts. These conduits were produced using a novel low pressure injection modeling process [63], [64], [65] which comprises a thermally induced phase transition (TIPS) process appropriate for the production of complex-shaped geometries. The mold design was flexible and allowed for the inexpensive production of variable numbers and diameters of channels, ranging from one 1.35 mm diameter channel to 100 0.08 mm channels. The channels provided an increased surface area for Schwann cell migration and adherence, as well as for axonal elongation.

The chosen polymer material was a high molecular weight 85:15 poly(dl-lactide-co-glycolide) (dl-PLGA). This material was selected as a model polymer system for demonstrating molding process feasibility. The process could be easily translated into other materials such as poly(l-lactide) or poly(caprolactone) polymers or poly(lactide-ε-caprolactone) and poly(trimethylene carbonate-co-ε-caprolactone) copolymers.